4-tert-butylpyrocatechol

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 April 2020 to 01 July 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Remarks:

Extended one-generation study (developmental results).

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 April 2020 to 01 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 443

Version / remarks:

June, 2018.

Deviations:

yes

Remarks:

The achieved dietary concentrations of 4-tert-butylpyrocatechol were not always within the acceptance criterion of 80 – 120 %.

GLP compliance:

yes

Specific details on test material used for the study:

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Details on mating procedure:

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Duration of treatment / exposure:

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Frequency of treatment:

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Duration of test:

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

300 ppm (nominal)

Remarks:

Anticipated dose level: 30 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 11-14 mg/kg/day for males and 14-17 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 19 mg/kg/day for males and 20 mg/kg/day for females.

Dose / conc.:

1 000 ppm (nominal)

Remarks:

Anticipated dose level: 100 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 42-50 mg/kg/day for males and 51-59 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 65 mg/kg/day for males and 68 mg/kg/day for females.

Dose / conc.:

3 125 ppm (nominal)

Remarks:

Anticipated dose level: 300 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 144-172 mg/kg/day for males and 176-194 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 221mg/kg/day for males and 230 mg/kg/day for females.

No. of animals per sex per dose:

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Control animals:

yes, plain diet

Details on study design:

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: At least once daily, beginning during the first administration of the test item and lasting throughout the treatment periods up to the day prior to necropsy.

BODY WEIGHT: Yes.
- Time schedule for examinations: On the first day of treatment (prior to administration), and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.

POST-MORTEM EXAMINATIONS: Yes.

Other: Parturition and maternal care.

For more information, see data detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River 2021 described in the section 7.8.1 Toxicity to reproduction.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes.
Examinations included:
- Gravid uterus weight: Yes.
- Number of corpora lutea: No.
- Number of implantations: Yes.
- Number of early resorptions: Yes.
- Number of late resorptions: Yes.

Blood sampling:

On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation, between 7.00 and 10.30 a.m. in the necropsy room, and samples were pooled per litter. If available, blood was collected from one male and one female pup per litter.
If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. When the target volume of 0.5 mL was not reached by pooling two pups, a third surplus pup of the same litter was added.

Thyroid hormones were evaluated (T4 and TSH).

Fetal examinations:

EXAMINATION OF PUPS:
- External examinations: Yes:
Culled pups (PND 4): Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.
- Soft tissue examinations: No.
- Skeletal examinations: No.
- Head examinations: No.
- Anogenital distance of all live rodent pups: yes.

Statistics:

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Indices:

Calculation of :
- Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) * 100.
- Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) * 100.
- Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) * 100.
- Percentage live males at first litter check (%): Number of live male pups at first litter check/Number of live pups at first litter check) * 100.
- Percentage live females at first litter check (%): Number of live female pups at first litter check/Number of live pups at first litter check) * 100.
- Viability index (%): (Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering) *100.
- Weaning index (%): (Number of live offspring on Day 21 after littering/Number of live offspring on Day 4 (after culling)) * 100.
- Percentage live males at weaning (%): (Number of live male pups on day 21 after littering/Number of live pups on day 21 after littering) * 100.
- Percentage live females at weaning (%): (Number of live female pups on day 21 after littering/Number of live pups on day 21 after littering) * 100.


The following parameters were examined:
Duration of gestation (number of days between confirmation of mating and the beginning of parturition), litter size, total number of offspring born, number of
live offspring on Day 1 after littering, number of live offspring on day 4 (before culling), number of live offspring on day 4 (after culling), number of live
offspring on day 21 after littering.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Gross pathological findings:

no effects observed

Description (incidence and severity):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.

Other effects:

no effects observed

Description (incidence and severity):

Parturition/Maternal care:
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Number of abortions:

no effects observed

Description (incidence and severity):

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Post-implantation loss:
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 93, 95 and 94% for the
control, 300, 1000 and 3125 ppm groups, respectively.

See table 1 and 2 in the field below 'Attached background material'.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Litter size was considered not affected by treatment with the test item.
Live litter sizes were 10.6, 11.4, 11.1 and 10.3 living pups/litter for the control, 300, 1000 and 3125 ppm groups, respectively.

See table 2 in the field below 'Attached background material'.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Duration of gestation was considered not to be affected by treatment with the test item.
See table 2 in the field below 'Attached background material'.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Gestation index was considered not to be affected by treatment with the test item. Except for one female at 1000 ppm (No. 173), that had a total litter loss, all pregnant females had live offspring on Day 1 of lactation. The gestation indices were 100% for the control, 300 and 3125 ppm groups and 95% for the 1000 ppm group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 125 ppm (nominal)

Based on:

test mat.

Remarks on result:

other:

Remarks:

No adverse maternal toxicity.

Dose descriptor:

LOEL

Effect level:

ca. 1 000 ppm (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Reduction of body weight and body weight gain in males and females at 3125 ppm (not considered to be adverse since these effects on body weight were minimals (less than 10%). Few hematology or clinical biochemistry parameters were affected by treatment with the test item at 1000 and 3125 ppm. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology. Findings in forestomach at 3125 ppm (hyperkeratosis and hyperplasia). Not considered as adverse.

Dose descriptor:

NOEL

Effect level:

ca. 300 ppm (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Reduction of body weight and body weight gain in males and females at 3125 ppm (not considered to be adverse since these effects on body weight were minimals (less than 10%). Few hematology or clinical biochemistry parameters were affected by treatment with the test item at 1000 and 3125 ppm. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology. Findings in forestomach at 3125 ppm (hyperkeratosis and hyperplasia). Not considered as adverse.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups during lactation:
Body weights of pups were considered not to be affected by treatment with the test item.
The higher mean pup weights on PND 1 (not statistically significant for males) were considered unrelated to the test item as the opposite effect (i.e. an decrease) would be expected in case of toxicity and as pup body weights were similar to concurrent controls on subsequent intervals.
See table 3 in the field below 'Attached background material'.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- Litter size:
Litter size was considered not affected by treatment with the test item.
Live litter sizes were 10.6, 11.4, 11.1 and 10.3 living pups/litter for the control, 300, 1000 and 3125 ppm groups, respectively.
See table 2 in the field below 'Attached background material'.

- Live Birth Index:
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment with the
test item.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment with the
test item. The live birth indices were 97, 100, 94 and 100% for the control, 300, 1000 and 3125 ppm groups, respectively.
The lower live birth index at 1000 ppm was mostly attributed to the total litter loss of Female No. 173 (11 dead pups in total). Additionally, 6 pups of the control group (Litter No. 125), 1 pup at 300 ppm (Litter No. 134) and 3 pups at 1000 ppm (Litter No. 164) were found dead at first litter check. No toxicological
relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered
normal for pups of this age.
See table 2 in the field below 'Attached background material'.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment with the test item.
For 2 pups from Litter No. 173 (1000 ppm), sex could not be determined due to cannibalism and were therefore assigned at random as males.
See table 2 in the field below 'Attached background material'.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- Viability Index:
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered to be unaffected by
treatment with the test item.
Viability indices (number of live offspring on PND 4 before culling as a percentage of total number of offspring on day 1 after littering) were 99% for the control,
1000 and 3125 ppm groups, and 98% for the 300 ppm group.

Two pups of the control group (one of Litter Nos. 103 and 121 each), 5 pups at 300 ppm (one of Litter Nos. 128, 138 and 146 each and two of Litter No. 136), 3 pups at 1000 ppm (two of Litter No. 159 and one of Litter No. 171) and 2 pups at 3125 ppm (one of Litter Nos. 193 and 199 each) were found dead or missing on
PND 2-4. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not
show a dose-related trend and remained within the range considered normal for pups of this age.
In addition, the last pup of Litter No. 125 (control) was missing on PND 2, resulting in a total litter loss for this dam.

See table 2 in the field below 'Attached background material'.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.
See table 4 in the field below 'Attached background material'.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

- The number of live pups offspring on day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered to be unaffected by
treatment with the test item (see field above for details 'Changes in litter size and weights').
- Weaning index: The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be
affected by treatment with the test item. The weaning indices were 99% for the control and 3125 ppm groups, and 100% for the 300 and 1000 groups.
Two pups of the control group (one of Litter Nos. 103 and 113 each) and 1 pup at 3125 ppm (Litter No. 177), were sacrificed in extremis or
found dead on PND 7, 18 or 17, respectively. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a
dose-related trend and remained within the range considered normal for pups of this age.

See table 2 in the field below 'Attached background material'.

External malformations:

no effects observed

Description (incidence and severity):

- Clinical signs during lactation (F1-Pups): No clinical signs occurred among pups that were considered to be related to treatment with the test item.
- Macroscopy until weaning (F1-Pups): No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be
related to treatment with the test item. For the pup of Litter No. 113 (control) that was found dead on PND 18, the stomach was noted to be grown together with
the pancreas at necropsy. Additionally, for the majority of the pup(s) of Female Nos. 125 (control), 134 (300 ppm), 164, 171, 173 (1000 ppm) that were
found dead at first litter check, beginning autolysis and/or absence of milk in the stomach were noted. For some pups cannibalism was observed. The nature and incidence of these and other macroscopic findings remained within the range considered normal for pups of this age or occurred in the control group only, and were therefore considered unrelated to the test item.
- Histopathology PND 4 F1-Pups: There were no test item-related microscopic findings in the stomach of the selected PND 4 pups. The only recorded finding was a cyst in the stomach of a control male pup.


See table 5 in the field below 'Attached background material'.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones: Serum T4 levels in male and female pups culled at PND 4 were considered not to be affected by treatment with the test item. It should be noted that for several animals within each group, values of 5.0 ng/mL were reported (i.e. ½ LOQ) as the original value was below LOQ, which is represented by
relatively low mean values. If the 5.0 ng/mL values would not be taken into account, mean values would be 12.6, 12.9, 12.9 en 11.9 ng/mL for control, 300, 1000 and 3125 ppm groups, respectively.

Note to table: Serum T4 levels of PND 4 pups are reported as females, but these were derived from pooled samples collected from male and female pups.

See table 6 in the field below 'Attached background material'.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 125 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other:

Remarks:

No effect on fetuses and on pups.

Conclusions:

- This extended one generation showed that TBC induced mean body weight gain reduction in males and females F0-generation animals in presence of TBC at
3125 ppm, resulting in a lower mean body weight during the pre-mating and mating period and in males F1-generation males at 1000 and 3125 ppm, resulting in a
lower mean body weight throughout the treatment period.
- At 3125 ppm (the higher tested dose), test item-related findings were present in the non-glandular stomach (forestomach) in adult rats. These test item related findings consisted of an increased incidence and severity of diffuse hyperkeratosis with or without squamous cell hyperplasia. Similar effects were observed to the F1-generation animal non-glandular stomach at 3125 ppm. The glandular stomach of F1-rats of Cohort 1A showed a higher incidence and severity of squamous cell hyperplasia compared to the stomachs of the F0-generation at 3125 ppm.

Based on this study, no general, reproductive or developmental toxicity was observed up to the highest dose tested (3125 ppm).

Executive summary:

The objective of this study was to provide an evaluation of the pre- and postnatal effects of 4-tert-butylpyrocatechol on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of 4-tert-butylpyrocatechol on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation. The dose levels in this study were selected to be 0, 300, 1000 and 3125 ppm, based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with dietary exposure of 4-tert-butylpyrocatechol in rats. The same dietary concentrations were used throughout the study, except for the lactation period. As food intake is considerably higher in lactating females, dietary concentrations were lowered for all treated groups (based on historical control data for relative food consumption) during the lactation period (PND 1-24). Chemical analyses of dietary preparations were conducted at nine occasions during the study to assess accuracy and homogeneity.

For the F0-generation, the following parameters and endpoints were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. For the F1-generation, the following parameters and endpoints were evaluated: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, estrous cycle, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined for the F0-generation: mating and fertility indices, precoital time, estrous cycle determination, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones) and histopathological examination of the stomach (PND 4 and PND 22-24 pups).

Results: Analysis of diet preparations confirmed thatthe test itemwas homogeneously distributed in the diet. The achieved dietary concentrations of 4-tert-butylpyrocatechol were however not always within the acceptance criterion of 80 – 120 %. A thorough check of the diet preparation procedures and analytical methodology revealed no explanation for the lower accuracies. In order to determine the worst case scenario exposure, test item intake was corrected for the mean achieved dietary concentrations per study period.

No mortality occurred during the study period that was considered to be related to treatment with the test item. In the F0-generation, one female of the control and 1000 ppm groups each, were euthanized on Lactation Day 2 or 1, respectively, as these females had a total litter loss. In the F1-generation, one male (1000 ppm, Cohort 1A) was sacrificed 5 days post weaning as it was assigned to the females.

No general, reproductive, or developmental toxicity was observed up to the highest dose tested (3125 ppm).

The following test item-related, non-adverse, effects were observed:

Parental results: Mean body weight gain was reduced in males and females at 3125 ppm, resulting in a lower mean body weight during the pre-mating and mating period. For females at 3125 ppm, no relevant differences were noted during the gestation period, whereas a higher mean body weight gain was observed from Day 7 of lactation onwards resulting in a slight recovery in mean body weight. Mean terminal body weight was 9 and 4% lower than concurrent control mean for males and females, respectively. As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse.

In animals at 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected by treatment with the test item, including decreased platelets counts in females, and decreased mean bile acid and creatinine concentrations in males. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology and as such they were regarded as non-adverse. In addition, mean serum levels oft hyroid stimulating hormone (TSH) were dose-dependently increased for females at all dose levels. As individual values generally remained within the historical control range, the change in serum TSH in the females was considered not to be adverse. 

At 3125 ppm, test item-related findings were present in the non-glandular stomach (forestomach) in adult rats. These test item-related findings consisted of an increased incidence and severity of diffuse hyperkeratosis (up to slight degree) with or without squamous cell hyperplasia.Since the recorded severities of these findings in adults remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, the combination of these findings was considered as a local, non-adverse test item-related effect.

Developmental results: Mean serum levels of TSH were decreased for male and female PND 22-24 pups (Cohort Surplus) at 3125 ppm. As individual values remained within the historical control range, the change in serum TSH was considered not to be adverse. 

F1-generation results: Mean body weight gain was reduced in males at 1000 and 3125 ppm, resulting in a lower mean body weight throughout the treatment period. Mean terminal body weights of Cohort 1A and 1B animals were 8 and 1% lower than control for males at 1000 ppm, and 9 and 6% lower than control for males at 3125 ppm, respectively. As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse.

In animals at 300, 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected, including increased mean lymphocyte counts and as a result increased mean total white blood cell count and mean large unstained cell count in both sexes, and decreased mean total bilirubin concentration in males. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with adverse anatomic pathology and as such they were regarded as non-adverse.

Similar to the F0-generation, test item-related findings were present in the non-glandular stomach at 3125 ppm. These test item‑related findings consisted of an increased incidence and severity of diffuse hyperkeratosis (up to slight degree) with or withoutsquamous cell hyperplasia.The glandular stomach of F1-rats of Cohort 1A showed a higher incidence and severity of squamous cell hyperplasia (incidence of 50% in males and females 60%, up to slight degree) compared to the stomachs of the F0-generation at 3125 ppm (incidence of 20% in males and 8% in females, at minimal degree). Furthermore, there were focal erosions (minimal degree) in the non-glandular stomach in 2/20 F1-rats of both sexes at 3125 ppm. Since the recorded severities of these findings remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, the combination of these findings was considered as a local, non-adverse test item-related effect.

In conclusion, based on the results of this extended one-generation reproductive toxicity study (including Cohorts 1), the following No Observed Adverse Effect Levels (NOAELs) of 4-tert-butylpyrocatechol were established:

General toxicity NOAEL:     

F0-generation: at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 172-194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose(3125 ppm).
F1-generation: at least 3125 ppm (on average corresponding to a corrected test article intake of 221 and 230 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).

Reproduction NOAEL: at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 176-194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).

Developmental NOAEL: at least 3125 ppm (on average corresponding to a corrected test article intake of 144 -172 and 176 -194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).

The test item (4 -tert-butylpyrocatechol) did not induce reprotoxic effects neither developmental effects up to the highest tested dose (3125 ppm)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

June, 2018.

Deviations:

yes

Remarks:

The achieved dietary concentrations of 4-tert-butylpyrocatechol were not always within the acceptance criterion of 80 – 120 %.

GLP compliance:

yes

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS::

- Premating exposure duration for parental (P0) animals : 10 weeks prior to mating.
- Basis for dose level selection :The dose levels were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test).
- Inclusion/exclusion of extension of Cohort 1B: No extension of the Cohort 1B. The substance is not wide dispersive, there is no exposure to consumers and clear conclusion on reprotoxicity can be done based on this study without F2 generation.
- Termination time for F2 : Not applicable.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : No inclusion of Cohorts 2A and 2B since the substance does not induce neurotoxic effect.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: No inclusion of Cohort 3 since the substance does not induce immunotoxic effect.
- Route of administration : The oral route of administration via dietary inclusion was selected since it is a common route of administration, which is recommended by the Regulatory Authorities for this type of study and as administration via oral gavage resulted in high toxicity due to corrosivity of the test item.

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butylpyrocatechol

EC Number:

202-653-9

EC Name:

4-tert-butylpyrocatechol

Cas Number:

98-29-3

Molecular formula:

C10H14O2

IUPAC Name:

4-tert-butylbenzene-1,2-diol

Test material form:

solid

Details on test material:

CAS N°: 98-29-3
CE N°: 202-653-9.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- 4-tert-butylpyrocatechol (CAS N° 98-29-3).
- Source: manufacturer.
- Purity: higher than 98%.
- No correction needed.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light in tightly closed containers.
- Stability and homogeneity of the test material in the diet under test conditions and during storage: (1) Stability in diet: Stability for at least 8 days in the freezer (closed containers) followed by 1 day at room temperature under normal laboratory light conditions (open containers) is confirmed over the concentration range 100 to 15000 ppm, Test Facility Study n° 20224318. Stability for at least 3 weeks in the freezer (≤-15°C) under normal laboratory light conditions is confirmed over the concentration range 100 to 15000 ppm, Test Facility Study n° 20224318. (2) Homogeneity analysis: Duplicate sets of samples (approximately 5 g accurately weighed) for each sampling time point were used for homogeneity analysis, the remaining samples were retained at the Test Facility as backup samples. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%. After acceptance of the analytical results, backup samples were discarded.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Stability in diet: Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20224318) demonstrated that the test item was stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20224318.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han).

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing and for reproduction and developmental toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive, and toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P-Generation: At initiation of administration, animals were 6 -7 weeks old.
- Weight at study initiation: P-Generation: At initiation of administration, animals weighed between 173 and 219 g (males) and between 111 to 146 g (females).
- Fasting period before study: No.
- Housing: On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV; 60x33x18 cm or type 2000P; 61x43.5x21.5 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; 38x22x18 cm). During the post-mating phase, males were housed in Macrolon type IV or type 2000P cages (61x43.5x21.5 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, 38x22x18 cm). During the lactation phase, females will be housed in Macrolon plastic cages (type III, 38x22x18 cm). Pups were housed with the dam until termination (unscheduled deaths, spares, and pups of Cohort Surplus) or until weaning on PND 21 (Cohorts 1A, 1B, 1C).
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The housing conditions were maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. Animals were separated during designated procedures/activities. Each cage were clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet (e.g. ad libitum): Prepared powder diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals were free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). The feed (without the test item) was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Animal identification: Prior to start of the treatment period, each F0-animal was identified using a chip. F1-pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet. From weaning (PND 21) onwards, the animals of Cohorts 1A, 1B and 1C were identified using a chip, which was implanted between PND 18-20. The animals of Cohort Surplus were identified by tail mark.
- Acclimation period: The F0-animals were allowed to acclimate to the Test Facility toxicology accommodation for 14 days before the commencement of administration.
- Animal enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities. Results of analysis for contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants that would interfere with the objectives of the study.
- Veterinary care: Veterinary care was available throughout the course of the study and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments were documented in the study records. A health inspection was performed before the initiation of administration.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The actual daily mean temperature during the study period was 20 to 22°C (target temperatures 18 to 24°C).
- Humidity (%): The actual daily mean relative humidity during the study was 44 to 75% (target humidity 40 to 70%). The values that were outside the targeted range occurred occasionally for 20 days with a maximum of 75% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained.

IN-LIFE DATES: From: 06 May 2020 to 27 November 2020

Administration / exposure

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
The test item was dissolved in acetone and pre-mixed with a small amount (~1-2 kg) of powder feed for one hour (± 5 minutes). An equal amount of acetone was used for all groups (Groups 1-4). The amount of acetone was based on the amount of test item used in the highest dose group (Group 4, 1:1 ratio w/w). Subsequently, this pre-mix was mixed for approximately 10 minutes with the remaining required amount of powder diet. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Diets were prepared at least biweekly for use at room temperature for a maximum of 1 day. If not used on the day of preparation, prepared diets were kept in the freezer (≤-15ºC) for a maximum of 3 weeks prior to use (stability for 3 weeks in the freezer (≤- 15°C)) was confirmed under Test Facility Study No. 20224318). Any remaining food left after filling the food hoppers may be stored at room temperature for a maximum of 1 day for supplementing food during the respective food consumption measurement interval.

The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout one specified phase of the study period. After termination, the actual test item intake has been estimated based on the body weight and food consumption values. The same diets remain in the food hopper for a maximum of one day. On the day of weighing the remaining diet in the food hopper, diet was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.

Details on mating procedure:

- M/F ratio per cage: Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating.
- Length of cohabitation: A maximum of 14 days was allowed for mating, after which the female that had not shown evidence of mating (No. 170) was separated from the cohabitated male.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
Once mating has occurred, the males and females were separated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample Collection and Analysis:
Diet preparation samples were collected for analysis as indicated below:

Food batches prepared for week 1 of treatment (a) (08 May 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 3 of treatment (a) (19 May 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 6 of treatment (a) (11 Jun 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 11 of treatment (a) (15 Jul 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 14 of treatment (a) (05 Aug 2020) (C): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 15 of treatment (a) (11 Aug 2020) (d): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 16 of treatment (a) (18 Aug 2020) (e) : All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 21 of treatment (a) (23 Sep 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for Week 25 of treatment (a) (28 Oct 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*

(a) The food batches prepared for these respective periods (Week X of treatment), analysis date included.
* The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations will be averaged and utilized as the concentration
results.
(c) Diets prepared for lactation period ‘Days 1-3’ of F0-females.
(d) Diets prepared for lactation period ‘Days 4-6’ of F0-females.
(e) Diets prepared for lactation period ‘from Day 7 onwards’ of F0-females.

All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility. For samples from diets prepared for Week 3 of treatment, UVVis spectra from 200-900 nm were recorded for one sample of each group. The spectra were
recorded after extraction, filtration and further dilution if required. These spectra were compared with spectra for blank extraction solvent and with spectra
for two calibration solutions. Residual samples were discarded after completion of the sample analysis.

Analyses described below were performed using a validated analytical procedure (Test Facility Study No. 20224318):
Concentration analysis: Duplicate sets of samples (approximately 5 g accurately weighed) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample
concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were
discarded.

Duration of treatment / exposure:

The duration of treatment period was the following:

- F0 males: 11 to 12 weeks (including 10 weeks pre-mating).
- F0 females: 16 to 18 weeks (including 10 weeks pre-mating).
- F0 females which failed to deliver or had a total litter loss: 13 to 15 weeks.

- F1 animals (Cohort 1A): 10 to 11 weeks.
- F1 animals (Cohort 1B): 12 to 13 weeks.
- F1 animals (Cohort 1C): 4 to 6 weeks.
- F1 animals (Cohort Surplus): not applicable.

Frequency of treatment:

Daily.

Details on study schedule:

- Age at mating of the mated animals in the study: [...] weeks

SELECTION, ASSIGNMENT, REPLACEMENT AND DISPOSITION OF ANIMALS:
F0-Animals were randomly assigned to groups at arrival. Males and females were randomized separately. Animals in poor health were not assigned to groups. At least upon receipt of the animals, a health inspection was performed and any assigned animals considered unsuitable for use in the study was replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
After initiation of administration, study animals may be replaced during the replacement period with alternate animals in the event of accidental injury, non-test item-related health issues, or similar circumstances. The alternate animals may be used as replacements on the study within 1 to 3 days. The disposition of all animals were documented in the study records.
On PND 4, eight pups from each litter of equal sex distribution (if possible) were selected to reduce variability among the litters. The non-selected pups were culled on PND 4.
- Selection of F1 generation:
On PND 21, pups from available litters per group were selected and assigned according to the following schedule. In total, 20 females with 8 live pups/litter were selected (if possible). Litters were selected and approved by the Study Director as per Test Facility SOPs. The selected litter numbers were the following:
- Cohort 1A (Reproductive toxicity): 20 males and 20 females. Age at necropsy: PND 89 - 95.
- Cohort 1B (Reproductive toxicity): 20 males and 20 females. Age at necropsy: higher or equal to PND 97.
- Cohort 1C (Reproductive toxicity): 20 males and 20 females. Age at necropsy: after vaginal patency/Balanopreputial separation positive.
- Surplus (Tyroid hormones): 10 males and 10 females (one pup (male or female) per litter and representative of 20 litters in total). Age at necropsy: PMD 22 - 24.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

300 ppm (nominal)

Remarks:

Anticipated dose level: 30 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 11-14 mg/kg/day for males and 14-17 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 19 mg/kg/day for males and 20 mg/kg/day for females.

Doses / concentrations 3

Dose / conc.:

1 000 ppm (nominal)

Remarks:

Anticipated dose level: 100 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 42-50 mg/kg/day for males and 51-59 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 65 mg/kg/day for males and 68 mg/kg/day for females.

Doses / concentrations 4

Dose / conc.:

3 125 ppm (nominal)

Remarks:

Anticipated dose level: 300 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 144-172 mg/kg/day for males and 176-194 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 221 mg/kg/day for males and 230 mg/kg/day for females.

No. of animals per sex per dose:

F0 animals: 25 animals per sex and per dose.

F1 animals:
- Cohort 1A: 20 animals per sex and per dose.
- Cohort 1B: 20 animals per sex and per dose.
- Cohort 1C: 20 animals per sex and per dose.
- Cohort Surplus: 10 animals per sex and per dose.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) (see DRF feed) with dietary exposure of 4-tert-butylpyrocatechol in rats and in an attempt to produce graded responses to the test item. During this study, Wistar Han rats received 0, 3125, 6250 and 12500 ppm by dietary administration for at least 28 days. No mortalities were observed during the treatment period. In the current study, the total duration of exposure was longer than in the preliminary reproductive toxicity study; there was be a 10-week premating period as opposed to the 2-week premating period. It was therefore expected that the effects on body weight and food consumption observed in the preliminary study may become more severe after a longer treatment period. In addition, during the current study, F1-animals were weaned at PND 21 and treated afterwards. Given the effects observed in the F0-animals and the pup body weights in the preliminary study, dose levels of 6250 and 12500 ppm were not considered appropriate for the current study. Considering this information, a dose level 3125 ppm was selected as high dose. Low and mid dose levels were selected to be 300 and 1000 ppm, respectively.

- Rationale for animal assignment (if not random): F0-Animals were randomly assigned to groups at arrival. Males and females were randomized separately.

- Fasting period before blood sampling for clinical biochemistry: The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0 animals and Cohort 1A animals housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.

- Administration of test materials:
The test item was administered to the appropriate animals by inclusion in the diet ad libitum. F0-males and females were treated up to and including the day before scheduled necropsy.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, spilled diet from the food hopper or when they commence eating for themselves.
From weaning onwards (PND 21), F1-animals of Cohorts 1A received diet containing test item up to and including the day before scheduled necropsy. The F1-animals of Cohort 1B and 1C received diet containing test item until the day of necropsy. The F1-animals of Cohort Surplus and Spares (not assigned to one of the cohorts) were housed with their dams until necropsy and therefore had access to the diet containing test item at the concentration provided to the dams.

The first day test diets containing test item were available to the animals was designated as Day 1.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout one specified phase of the study period. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
The same diets remained in the food hopper for a maximum of one day. Daily the remaining diet in the food hopper was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.

Examinations

Parental animals: Observations and examinations:

F0-GENERATION:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: At least twice daily, in the morning and at the end of the working day, throughout the study.
- Procedure: Animals were observed for general health/mortality and moribundity. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: At least once daily, beginning during the first administration of the test item and lasting throughout the treatment periods up to the day prior to necropsy.
- Procedure: Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected.

ARENA OBSERVATIONS: Yes.
- Time schedule: Once before the first administration of the test item and at weekly intervals during the treatment period.
- Procedure: Clinical observations were conducted in a standard arena.

BODY WEIGHT: Yes.
- Time schedule for examinations: Males and females were weighed individually on the first day of treatment (prior to administration), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 postcoitum and during lactation on PND 1, 4, 7, 14 and 21.
- Procedure: Animals were weighed individually. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.
- Time schedule and procedure: Food consumption of animals was quantitatively measured once daily throughout the study from Day 1 of administration onwards except for males and females which were housed together for mating and for females without evidence of mating. Measurements were conducted at approximately the same time.

WATER CONSUMPTION: Yes.
- Time schedule for examinations: Regular basis throughout the study.
- Procedure: Water consumption was monitored by visual inspection of the water bottles.

OTHER:
- Laboratory evaluations: F0-Animals (selected animals - 10/sex/group): The following parameters were examined: Hematology, coagulation, clinical chemistry, thyroid hormone and urinalysis. For more details and tables see the field below 'Any other information on materials and methods incl. tables'.
- General Reproduction data: From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. For more details see the field below 'Any other information on materials and methods incl. tables'.

Oestrous cyclicity (parental animals):

F0 GENERATION:

- Time schedule: Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was
observed. Vaginal lavage was continue for those females with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal lavage was also be taken to determine the stage of estrus.
- Procedure: Estrous stages were determined by examining the cytology of vaginal lavage samples.

Sperm parameters (parental animals):

F0 GENERATION:
For all males, the following assessments were performed:
- Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy.
- Sperm motility and progressive motility were assessed from all samples.
- Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal were recorded. Evaluation has been performed for all samples.
- One epididymis (right) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing, the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

Litter observations:

IN-LIFE PROCEDURES, OBSERVATIONS, AND MEASUREMENTS - F1-GENERATION UNTIL WEANING (PND 21):

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes.
Culling on PND 4. To reduce variability among the litters, eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, were not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) is acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring (pups until weaning (PND 21)):
- Mortality/moribundity: Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Pup 2 of Litter No. 103 was sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).
- Clinical observations: Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.
- Body weight: Live pups were weighed individually on PND 1, 4, 7, 13 and 21. For animals of Cohort Surplus, a terminal weight was recorded on the day of scheduled necropsy.
- Sex ratio: Sex was externally determined for all pups on PND 1, 4 and 13.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/Nipple retention: All male pups in each litter were examined for the number of areola/nipples on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
Stillborn pups and pups found dead between birth and PND 13 will be sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology.
If possible, defects or cause of death were evaluated.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No.
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No.

OTHER:
- Thyroid hormone. F1 culled pups (2 pups per litters). Time point(s): PND 4. For more details and tables see the field below 'Any other information on materials and methods incl. tables'.


IN-LIFE PROCEDURES, OBSERVATIONS, AND MEASUREMENTS - F1-GENERATION FROM WEANING (PND 21) ONWARDS:

F1 GENERATION FROM WEANING (PMD 21): Cohorts 1A; 1B and 1C (60 animals/sex/group):
The in-life procedures, observations, and measurements listed below were performed for all F1-animals from weaning (PND 21) onwards, except for the animals of Cohort Surplus and spare F1-animals as these were terminated on/before PND 24.

CAGE SIDE OBSERVATIONS: Yes, cohorts 1A, 1B and 1C.
- Time schedule: Twice daily throughout the study.
- Procedure: Animals were observed for general health/mortality and moribundity. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes, cohorts 1A, 1B and 1C.
- Time schedule: At least once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
- Procedure: Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade were predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) were scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected.

ARENA OBSERVATIONS: Yes, cohorts 1A, 1B and 1C.
- Time schedule: Once on the day of weaning and thereafter at weekly intervals during the treatment period.
- Procedure: Animals were observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs were recorded.

BODY WEIGHT: Yes, cohorts 1A, 1B and 1C.
- Time schedule for examinations: Weekly from weaning onwards. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. For animals of Cohorts 1A and 1B, a terminal weight was recorded on the day of scheduled necropsy.
- Procedure: Animals were individually weighed.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, cohorts 1A, 1B, 1C.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.
- Time schedule and procedure: Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy. For animals of Cohort 1A, food consumption was measured up to the day prior to scheduled necropsy. For animals of Cohorts 1B and 1C, food consumption was measured up to and including the day of necropsy.

WATER CONSUMPTION: Yes, cohorts 1A, 1B, 1C.
- Time schedule for examinations: Regular basis throughout the study.
- Procedure: Water consumption was monitored by visual inspection of the water bottles.

OTHER:
LABORATORY EVALUATIONS:
- Laboratory evaluations: F1-animals (cohort 1A animals - selected animals (10/sex/group)): The following parameters were examined: Hematology, coagulation, clinical chemistry, thyroid hormone and urinalysis. For more details and tables, see the field below 'Any other information on materials and methods incl. tables'. Time point(s): on the day of scheduled necropsy.
- Thyroid hormones for F1-animals - Cohorts Surplus (All surplus pups). For more details and tables, see the field below 'Any other information on materials and methods incl. tables'. Time point(s): PND 22-24.

VAGINAL PATENCY (Cohorts 1A, 1B and 1C):
- Time schedule: Daily for all females from PND 25 onwards until vaginal patency was present.
- Procedure: Vaginal patency (vaginal opening) was monitored by visual inspection of the vaginal area. Body weight was recorded on the day of acquisition of vaginal patency.

BALANOPREPUTIAL SEPARATION (Cohorts 1A, 1B and 1C):
- Time schedule: Daily for all males from PND 35 onwards, until balanopreputial separation was present.
- Procedure: Balanopreputial separation (prepuce opening) was monitored by visual inspection of the genital area. Body weight was recorded on the day of acquisition of balanopreputial separation.

STAGE OF ESTRUS DETERMINATION (Cohorts 1A, 1B and 1C):
- Time schedule: On the day of scheduled necropsy, a vaginal lavage was be taken.
- Procedure: Estrous stages were determined by examining the cytology of vaginal lavage samples.

ESTROUS CYCLE DETERMINATION (Cohort 1A only):
- Time schedule: During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88.
- Procedure: Both periods: Estrous stages were determined by examining the cytology of vaginal lavage samples.
The estrous cycle data of the first period is not reported. Data were retained in the raw data.

SPERM ANALYSIS (Cohort 1A only): For descriptions and tables, see the field below 'Any other information on materials and methods incl. tables'.

SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS (Cohort 1A only): For descriptions and table (table 18), see the field below 'Any other information on materials and methods incl. tables'.

Postmortem examinations (parental animals):

SACRIFICE:

F0 Generation:

Terminal procedures are summarized in the table 11 (see table 11 in the field below ' Anyother information on materials and methods inc. tables').

All animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.

Scheduled necropsies are summarized below:
- Males which sire: After successful mating and a minimum of 10 weeks of treatment.
- Males which failed to sire: At the end of the mating period and after a minimum of 10 weeks of treatment.
- Females which delivered: Lactation Day 23-25.
- Females which failed to deliver: With evidence of mating: Post-coitum Days 26-27 (N°108, 117, 124, 130, 141, 144, 151, 158, 184, 187, 192 and 195). Without evidence of mating: Approximately 27 days after the last day of the mating period (N° 170).
- Females with total litter loss (N° 125 and 173): within 24 hours after the last pup was found dead or missing.

Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available.

GROSS NECROPSY:
F0 Generation:
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.


ORGAN WEIGHTS
F0 Generation:
The organs identified for weighing in the Tissue Collection and Preservation table (see table 12 in the field below ' Anyother information on materials and methods inc. tables') were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.


HISTOLOGY / HISTOPATHOLOGY
Tissue collection and preservation:
Representative samples of the tissues identified in the Tissue Collection and Preservation table (see table 12 in the field below ' Anyother information on materials and methods inc. tables') were collected from all animals and preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.
For females which fail to deliver a complete litter, uterine contents (i.e. any fetuses, placenta and implantation sites) have to be fixed (if applicable), but not have to be examined histopathologically in first instance.
The tissues indicated in Table 12 (see field 'Anyother information on materials and methods incl. tables') were prepared for microscopic examination and weighed, respectively.
Tissues were processed at Charles River Laboratories Frederick. Tissues in the Tissue Collection and Preservation table (see table 12 in the field below ' Anyother information on materials and methods inc. tables') from a selection of the F0-animals identified in the Terminal Procedures table (see table 11) were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).
All tissues as defined under Histology from a selection of the F0-animals identified in the Terminal Procedures table (see table 12 in the field below ' Anyother information on materials and methods inc. tables) were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation will be communicated to the Study Director; tissues will be evaluated and reported. Any additional stains or evaluations, if deemed necessary by the pathologist, will be added by study plan amendment following discussion with the Study Director and in consultation with the Sponsor.

Terminal procedures, tissue collection/preservation and organ weights for F0-animals are summarized in the field below 'Any other information on materials and methods incl. tables', in tables 11 and 12.

Postmortem examinations (offspring):

TERMINAL PROCEDURES - F1-GENERATION UNTIL WEANING:
- Unscheduled deaths (F1-Generation):
The pup sacrificed in extremis on PND 7 was euthanized by an intraperitoneal injection of sodium pentobarbital.
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally, if possible).
Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

- Culled Pups (PND 4) (F1 Generation):
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. From two extra pups per litter, blood were collected for the F1-generation, if possible.
Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.
In addition, the stomach was collected and preserved in 10% buffered formalin at necropsy for 10 selected animals/sex/group. These animals were selected and approved in advance by the Study Director in the study files. Histology and histopathology were performed on all collected stomachs.

TERMINAL PROCEDURES - F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C, cohort suplus and Spare F1-animals):
Terminal procedures for F1 - generation are summarized in the table 13 (See the field below 'Any other information on materials and methods incl. tables').

SACRIFICE:
Spare F1-animals which are not assigned to one of the Cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital. Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally). Descriptions of all external abnormalities were recorded.
For all animals, necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. Tissues were preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

GROSS NECROPSY:
COHORT 1A:
Scheduled necropsy of Cohort 1A was conducted on PND 89-95. Cohort 1A animals surviving to scheduled necropsy were deprived of food overnight (with a maximum of 24 hours) before necropsy, but water was available. The animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. For data collection and preservation, see table 14 in the field below 'Any other information on materials and methods incl. tables'.

COHORT 1B:
Scheduled necropsy of Cohort 1B was conducted on ≥ PND 97. Cohort 1B animals were not deprived of food overnight before necropsy. These animals were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. For data collection and preservation, see table 15 in the field below 'Any other information on materials and methods incl. tables'.

COHORT 1C:
Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy and no terminal body weight was recorded.
The animals were deeply anesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. For data collection and preservation, see table 16 in the field below 'Any other information on materials and methods incl. tables'.

COHORT SURPLUS:
Scheduled necropsy of Cohort Surplus was conducted on PND 22. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. For data collection and preservation, see table 17 in the field below 'Any other information on materials and methods incl. tables'.
On PND 22, blood samples (1.0 mL) were collected between 8.00 and 11.30 a.m. from all animals by aorta puncture under anesthesia using isoflurane as part of the necropsy procedure. Blood samples were collected into serum tubes for measurement of thyroid-stimulating hormone (TSH) and thyroxine (T4).

ORGAN WEIGHT:
COHORT 1A:
The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table (see table 14 in the field below 'Any other information on materials and methods incl. tables') were weighed and collected.

COHORT 1B:
The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table (see table 15 in the field below 'Any other information on materials and methods incl. tables') were weighed and collected.

COHORT 1C:
No organ weights for this cohort.

COHORT SURPLUS:
The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table (see table 17 in the field below 'Any other information on materials and methods incl. tables') were weighed and collected.

HISTOLGY / HISTOPATHOLOGY:
Histology: Tissues were processed at Charles River Laboratories Frederick. Tissues in the Tissue Collection and Preservation tables (see tables 14 to 17 in the field 'Any other information on materials and methods incl. tables') from a selection of the F1-animals identified in the Terminal Procedures tables (see table 13) were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).
Histopathology: All tissues as defined under Histology from a selection of the F1-animals identified in the Terminal Procedures tables (see stables 14 to 17) were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

A peer review on the histopathology data was performed by a second pathologist, during which full tissue review was performed of at least five animals/sex/group of the highest dose with 50% or greater survival/sex. Remaining tissues were reviewed according to standard operating procedures.
Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported


COHORT 1A:
In addition to the procedures described above, for Cohort 1A animals of Groups 1 and 4, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the cohort 1A animals of group 1 and 4 for quantitative evaluation of follicles (primordial and small growing follicles counted together), as well as corpora lutea.

Statistics:

STATIISTICS
DATA COLLECTION IN TOXDATA
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1or 5% levels. Numerical data collected on scheduled occasions were analyzed according to sex and occasion. Descriptive statistics number, mean and
standard deviation were reported when possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate.
The following pairwise comparisons were made:
Group 2 vs Group 1
Group 3 vs Group 1
Group 4 vs Group 1.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test)
Non-parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test)
Incidence: An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenthe overall test is significant.

DATA COLLECTED IN PROVANTIS
Inferential methods: All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided
tests and were reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest are the following:
Group 2 vs Group 1
Group 3 vs Group 1
Group 4 vs. Group 1
Parametric/non-Parametric method was used for analysis of Hematology, Coagulation and Clinical variables.Parametric/non-Parametric: Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons will be conducted usingDunnett’s or Dunn’s test, respectively.
Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Reproductive indices:

Reproduction variables:

For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual
values of F0-females, the remaining group values were calculated from the total number in each group.

- Mating index males (%) = Number of males mated / Number of males paired * 100.

- Mating index females (%) = Number of females mated / Number of females paired * 100.

- Precoital time = number of days between initiation of cohabitation and confirmation of mating.

- Fertility index males (%) = Number of pregnent females / Number of males mated * 100.

- Fertility index females (%) = Number of pregnent females / Number of females mated * 100.

- Gestion index (%) = Number of females with living pups on Day 1/ Number of pregnant females.

- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition.

Offspring viability indices:

Developmental variables:

For each group, the following calculations were performed:

- Post-implantation survival index (%) = Total number of offspring born / Total number of uterine implantation sites * 100.
(Post-implantation survival index will be expressed as 100% when the number of offspring exceeds the number of implantation sites recorded).

- Live birth index (%) = Number of live offspring on day 1 after littering / Total number of offspring born * 100.

- Percentage live males at first litter check (%) = Number of live male pups at first litter check / Number of live pups at first litter check * 100.

- Precentage live females at first litter check = Number of live female pups at first litter check / Number of live pups at first litter check * 100.

- Viability index (%) = Number of live offspring on Day 4 before culling / Number of live offspring on Day 1 after littering * 100.

- Weaning index (%) = Number of live offspring on Day 21 after littering / Number live offspring on day 4 (after culling) * 100.

- Percentage live males at weaning (%) = Number of live male pups on Day 21 after littering / Number of live pups on Day 21 after littering * 100.

- Percentage live females at weaning (%) = Number of live female pups on Day 21 after littering / Number of live pups on Day 21 after littering * 100.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

At 3125 ppm, one female had a greasy coat and red staining (i.e. chromodacryorrhoea) on the back during Week 9/10 of treatment, which recovered in early
week 10. Additionally, piloerection was observed for 5 females of the same group (group 4) (Nos. 180, 185, 191, 193 and 199) during week 15/16 of treatment. For most females, piloerection was noted on one or two days only. At the incidence observed and as clinical signs were transient, they were considered to be
unrelated to treatment with the test item.
Other clinical signs noted during the treatment period (i.e. scabs and alopecia) occurred within the range of background findings to be expected for rats of this
age and strain which are housed and treated under the conditions in this study and did not show any apparent dose related trend. At the incidence observed,
these were considered to be unrelated to treatment with the test item.

See table 23 in the field below 'Attached backgrowd material'.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No test item-related mortality occurred during the study period.
Two females, one of the control and 1000 ppm groups each (Nos. 125 and 173), were euthanized on Lactation Day 2 or 1, respectively, as these females had a
total litter loss.

See table 22 in the field below 'Attached backgrowd material'.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item-related differences in body weight were observed at 3125 ppm.

Mean body weight gain of males at 3125 ppm was lower from Day 8 of treatment onwards. This resulted in a lower mean body weight of these males during the pre mating and mating period (not always statistically significant). Mean terminal body weight was 9% lower than control mean (statistically significant).

Mean body weight gain of females at 3125 ppm was lower from Day 22 of treatment onwards up to and including Day 1 of the mating period. On Day 1 of the mating period, mean body weight was 4% lower than control mean. Mean body weight gain was similar between all groups during gestation, but the mean
body weight of these females remained statistically significantly lower throughout gestation. The difference in body weights between high dose and control
group was more or less constant (6-8% lower) and most likely related to the decreased body weights at the end of the pre mating period at 3125 ppm. During lactation, a higher mean body weight gain was observed for these females from Day 7 of lactation onwards, resulting in a slight recovery with regards to
mean body weight. On Day 1 of lactation, mean body weight was 9% lower than control mean, whereas on Day 21 of lactation the difference was reduced
to 5%. Mean terminal body weight of these females was 4% lower than control mean (not statistically significant).

Body weights and body weight gain of animals treated at 300 or 1000 ppm were considered to have been unaffected by treatment with the test item.

See table 24 in the field below 'Attached backgrowd material'.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption: No toxicologically relevant changes in food consumption before or after correction for body weight were noted. Mean absolute food
consumption of males at 3125 ppm was lower during weeks 4-8 of treatment and mean relative food consumption during weeks 4-7 of treatment (statistically significant). As no relevant differences were observed from week 8 of treatment onwards and the mean of means food consumption (absolute and relative) was similar to concurrent control, this temporary and minimal decrease in food consumption was considered not toxicologically relevant.
Mean absolute food consumption of females at 3125 ppm was slightly lower during the pre-mating and gestation periods (not always statistically significant). As relative food consumption was similar to concurrent control mean, this decrease was attributed to the lower body weights observed for these females
rather than a direct effect of the test item. No toxicologically relevant differences were observed in food consumption of these females during the lactation
period.
Food consumption before or after correction for body weight was considered unaffected by treatment with the test item up to 1000 ppm.

Any other statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment with
the test item since no trend was apparent regarding dose and duration of treatment.

Note: When compared to the pre-mating period, a generally higher food intake (absolute and relative to body weight) was measured in females during the
phases of gestation and lactation, with values for the control group within the normal range. The observed higher food intake is a normal physiological
process. It reflects the increased nutritional need of the dams during the periods of gestation and lactation. Furthermore, from the third week of lactation
onwards also their pups start to consume solid feed.

Test item intake: Mean test article intake over the study period was indicated in the table 26. Lower accuracies were measured for the prepared powder diet. In order to determine the worst case scenario exposure, the table 27 presents the mean of means test item intake corrected for the mean of means accuracy per period. See tables 26 and 27 in the field below 'Any other information on results incl. tables'.

See also table 25 in the field below 'Attached backgrowd material' for details on F0-generation food consumption.

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology:
The following changes distinguished treated females from control animals:
- Mean concentration of platelets was decreased at 1000 and 3125 ppm (0.92x and 0.89x of control, respectively; not statistically significant). Given the
magnitude or based on a dose-related response, this change was considered to be test item-related.
Hematological parameters of treated males were considered not to have been affected by treatment with the test item.
Mean concentrations of reticulocytes in treated males were lower than concurrent control mean. This was considered to be unrelated to the test item as it
was attributed to a higher control mean due to a relatively high value of Male No. 7.
Any other changes in hematology parameters were considered to be unrelated to administration of the test item due to the minimal magnitude of the change,
variation in direction of change, absence of a dose response, relatively high values in single animals and/or absence of biological relevance.
See table 28 in the field below 'Attached backgrowd material'.

Coagulation:
No toxicologically relevant changes were noted in coagulation parameters. The shorter prothrombin time (PT) of males at 3125 ppm was considered not to be of toxicological relevance given the minimal magnitude of the change.
See table 29 in the field below 'Attached backgrowd material'.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following changes distinguished treated from control animals:
- Mean bile acid concentration was decreased in males at 3125 ppm (0.61x of control; not statistically significant).
- Mean creatinine concentration was decreased in males at 3125 ppm (0.87x of control).
Given the magnitude, these changes were considered to be test item-related.

Mean total bilirubin concentration was decreased in males at 1000 and 3125 ppm (0.84x and 0.83x of control, respectively; not statistically significant at 1000
ppm). Mean inorganic phosphate concentration was increased in females at 3125 ppm (1.11x of control; not statistically significant). Given the magnitude, as
individual values generally remained within concurrent control range and/or in absence of a dose-related trend, these changes were considered not toxicologically relevant.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of
a dose-related trend.

See table 30 in the field below 'Attached backgrowd material'.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean TSH values of treated females were 1.22x, 1.58x and 2.52x of control at 300, 1000 and 3125 ppm, respectively (not statistically significant). The increased mean TSH value at 3125 ppm was partially caused by one female with a relatively high value. Without this female, the group mean would be 1.84x of control.
Other individual values generally remained within the concurrent control range as well as the historical control range (Historical control data of TSH (mU/L) in
female Wistar Han rats (2017-2021); Mean = 0.204, P5-P95 = 0.030-0.626 (n=116)).
Mean T4 values were also increased in treated females (not statistically significant), but this change was considered not to be of toxicological relevance as the
opposite effect (i.e. a decrease) would be expected in case of target organ toxicity and as no clear dose-response was observed.

For males, the mean T4 concentration was lower at 3125 ppm and the mean TSH concentration was higher at all dose levels when compared with concurrent
controls (all not statistically significant). As the decrease in mean T4 was minimal, the increase in mean TSH occurred in the absence of a dose-related response
and as individual values generally remained within concurrent control mean, no relationship with the test item was indicated.

See table 31 in the field below 'Attached backgrowd material'.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis parameters of treated rats were considered not to be affected by treatment with the test item.
For 1 and 3 males at 1000 and 3125 ppm, respectively, a small response (1+) was recorded for bilirubin while all control and 300 ppm were negative. At the
incidence and severity observed and as the positive result could not be confirmed, this difference was considered not test item-related.

See table 32 in the field below 'Attached backgrowd material'.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with 4-tert-butylpyrocatechol were noted in the stomach of the 3125 ppm group males and females and are summarized in the table 35 (See table 35 in the field below 'Any other information on materials and methods incl. tables').
Non-glandular stomach (i.e. forestomach): An increased incidence and/or severity of diffuse hyperkeratosis (up to slight degree) accompanied in a few animals by squamous cell hyperplasia (minimal degree) was present in both sexes at 3125 ppm.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no
test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

See also table 36 in the field below 'Attached backgrowd material'.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for control Female No. 106 (with normal litter) and Female No. 187 at 3125 ppm (not pregnant). Given their incidental nature and occurrence in the control group, these findings did not indicate a relation with the test item.

See table 37 in the field below 'Attached backgrowd material'.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Mean percentage of motile and progressive sperm were lower in treated males when compared with control. The percentage of motile sperm was 0.79, 0.82 and 0.88x of control and of progressive sperm 0.64, 0.68 and 0.79x of control for the 300, 1000 and 3125 ppm groups, respectively (not statistically significant for
percentage of motile sperm at 3125 ppm). All individual values (except for one male at 300 ppm) remained within the normal range for rats of this age and strain.
In absence of a dose-related response and given the absence of any reproductive/developmental toxicity, these differences were considered not toxicologically relevant.
Sperm concentration and morphology were considered to be unaffected by treatment with the test item.
The mean sperm count in males at 300 and 1000 ppm was increased (1.22 and 1.18x of control, respectively). Due to the direction of change and in the absence of a dose-related response, this difference was considered unrelated to the test item.

See table 38 in the field below 'Attached backgrowd material'.

Reproductive performance:

no effects observed

Description (incidence and severity):

- Mating Index: Mating index was considered not to be affected by treatment with the test item for both sexes. Except for one female at 1000 ppm (No. 170),
all females showed evidence of mating.
- Precoital Time: Precoital time was considered not to be affected by treatment with the test item. All females showed evidence of mating within 4 days, with
exception of one female (No. 180) at 3125 ppm, for which it took 13 days before mating could be confirmed.
- Number of Implantation Sites: Mean number of implantation sites at 3125 ppm was decreased (0.94x of control; not statistically significant). Based on the
magnitude of change and as individual values remained within concurrent control range, this difference was considered not toxicologically relevant. Number
of implantation sites was considered not to be affected by treatment with the test item up to 1000 ppm.
- Fertility Index: Fertility index was considered not to be affected by treatment with the test item. The fertility indices were 88% for the control, 300 and 1000
ppm groups, and 84% for the 3125 ppm groups for both sexes.
A total of 3 females from the control, 300 and 1000 ppm groups each and 4 females at 3125 ppm were not pregnant. Since these cases of non-pregnancy
showed no clear dose related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was considered not to be related to treatment with the test item.
- Histopathological evaluation of reproductive preformance: Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present. A total of 3 females from the control, 300 and 1000 ppm groups each and 4 females at 3125 ppm were not pregnant, despite evidence of mating. In addition, mating could not be confirmed for one couple in the 1000 ppm group. Furthermore, one female of the control group and one female at 1000 ppm had
total litter loss at Lactation Day 2 and 1, respectively. There were no microscopic findings in the reproductive organs (and mammary gland in the case of total
litter loss) which could account for this lack of healthy offspring. Since these cases of non-pregnancy and total litter loss showed no clear dose related
incidence across the dose groups and given the absence of any reproductive/developmental toxicity, this was considered not to be related to treatment with
the test item.

See table 39 in the field below 'Attached backgrowd material'.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 125 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect (general toxicity).

Remarks on result:

other:

Remarks:

General toxicity: Only non-adverse effects were observed.

2

Dose descriptor:

LOEL

Effect level:

ca. 1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

clinical biochemistry

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

General toxicity: Reduction of body weight and body weight gain in males and females at 3125 ppm (not considered to be adverse since these effects on body weight were minimals (less than 10%). Few hematology or clinical biochemistry parameters were affected by treatment with the test item at 1000 and 3125 ppm. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology. Findings in forestomach (local effects) at 3125 ppm (hyperkeratosis and hyperplasia), not considered as adverse.

3

Dose descriptor:

NOEL

Effect level:

ca. 300 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

clinical biochemistry

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

General toxicity: Reduction of body weight and body weight gain in males and females at 3125 ppm (not considered to be adverse since these effects on body weight were minimals (less than 10%). Few hematology or clinical biochemistry parameters were affected by treatment with the test item at 1000 and 3125 ppm. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology. Findings in forestomach (local effects) at 3125 ppm (hyperkeratosis and hyperplasia), not considered as adverse.

4

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 125 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive effect.

Remarks on result:

other:

Remarks:

Reproductive toxicity. No reproduction toxicity was observed up to the highest dose level tested (3125 ppm).

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1 - GENERATION - F1-PUPS DURING LACTATION:
No clinical signs occurred among pups that were considered to be related to treatment with the test item.
An abnormal posture of the left or right hindleg was observed for 2 control pups (one of Litter Nos. 107 and 123 each), 1 pup at 300 ppm (Litter No. 147),
1 pup at 1000 ppm (Litter No. 160) and 1 pup at 3125 ppm (Litter No. 196). This clinical sign was observed from PND 7, 20 or 13 onwards. Based on the
absence of a dose-response or and occurrence in the control group, the abnormal posture of one of the hindlegs was considered unrelated to the test item.
Fissures of the left foreleg were observed between PND 7-9 and missing eyes were observed from PND 17 onwards in 2 pups of the control group (both of
Litter No. 103). Additionally, another pup of this litter was sacrificed in extremis on Day 7 to prevent suffering due to the excess of Indian ink (used for
identification on PND 1) observed ventrally. Due to occurrence in the control group only, above mentioned clinical signs were unrelated to the test item.
The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore considered unrelated
to the test item.

See tables 40/41 in the field below 'Attached backgrowd material'.


F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Surplus):
No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Any clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
One Cohort 1C female of the control group (No. 540) was weaned while missing both eyes. Based on the body weight gain data, there was no impact on the development of this animal. As such, this clinical sign had no impact on animal welfare or the study results.

See table 47 in the field below 'Attached backgrowd material'.
Note for the table: “Day 1” of treatment is the day that the first animals were weaned. For those animals that were not yet weaned on the day that the first
animals were weaned, a “.” appears in the table.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1-GENERATION - OFFSPRINGS:
Viability index:
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered to be unaffected by
treatment with the test item.
Viability indices (number of live offspring on PND 4 before culling as a percentage of number of offspring on Day 1 after littering) were 99% for the control,
1000 and 3125 ppm groups, and 98% for the 300 ppm group.

Two pups of the control group (one of Litter Nos. 103 and 121 each), 5 pups at 300 ppm (one of Litter Nos. 128, 138 and 146 each and two of Litter No. 136), 3 pups at 1000 ppm (two of Litter No. 159 and one of Litter No. 171) and 2 pups at 3125 ppm (one of Litter Nos. 193 and 199 each) were found dead or missing on PND 2-4. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidencedid not show a dose-related trend and remained within the range considered normal for pups of this age.
In addition, the last pup of Litter No. 125 (control) was missing on PND 2, resulting in a total litter loss for this dam.

See tables 40/41 in the field below 'Attached backgrowd material'.


F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Surplus):
No mortality occurred during the study period that was related to treatment with the test item.
Animal No. 633 (1000 ppm, Cohort 1A) was sacrificed 5 days post weaning as it appeared to be male upon daily observations while it was assigned to the
females. This male was replaced by Female No. 673 (1000 ppm, Cohort 1C). All data of Animal No. 673 was reported under No. 633 and all data of Animal No. 633 was reported under No. 673 or No. 633-A.
For Animal Nos. 539, 736 and 750, the necropsy date was recorded as being the first date of weaning. These animal numbers were planned in the computer system, but were not used in the study as no animal was available at weaning.

See table 48 in the field below 'Attached backgrowd material'.
Note: The date under “Treatment from” in Table 48 is the date that the first animals reached PND 21. Actual treatment of the animals commenced on the date
that the respective animals reached PND 21.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1-GENERATION - F1-PUPS - DURING LACTATION:
Body weights of pups were considered not to be affected by treatment with the test item.
The higher mean pup weights on PND 1 (not statistically significant for males) were considered unrelated to the test item as the opposite effect (i.e. an
decrease) would be expected in case of toxicity and as pup body weights were similar to concurrent controls on subsequent intervals.

See table 42 in the field below 'Attached backgrowd material'.

F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Surplus):
Test item-related differences in body weight were observed at 1000 and 3125 ppm.
Mean body weight gain of males at 1000 and 3125 ppm was lower from Day 15 of treatment onwards, which resulted in a lower mean body weight of these males throughout the treatment period (not always statistically significant). Mean terminal body weights of Cohort 1A and 1B animals were 8 and 1% lower
than control for males at 1000 ppm and 9 and 6% lower than control for males at 3125 ppm, respectively (not statistically significant for Cohort 1B).
Mean body weight gain of females at 3125 ppm was slightly lower from Day 36 of treatment onwards (not statistically significant). No clear differences were observed in mean body weight during the treatment period, but terminal body weight of these females was 5 and 1% lower than control mean for Cohort 1A
and 1B animals, respectively (both not statistically significant). Given the magnitude of change, the observed differences were considered not toxicologically relevant.
Body weights and body weight gain of males treated at 300 and females treated at 300 or 1000 ppm were considered to have been unaffected by treatment with the test item.

Note: “Day 1” of treatment is the day that the first animals were weaned. Body weight of animals that were weaned during the subsequent 7 days were all
entered under that day. Body weight of animals that were weaned in the subsequent week were all entered under “Day 8”. This may have resulted in the
statistically significant changes as described above. Since the majority of animals were weaned within 7 days after commencement of weaning of the first
animals, “Day 8” was used as the reference day for body weight gain calculations.

See table 49 in the field below 'Attached backgrowd material'.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Surplus):
No toxicologically relevant changes in food consumption before or after correction for body weight were noted.
Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment with the test item since no trend was apparent regarding dose and duration of treatment.

See table 50 in the field below 'Attached backgrowd material'.

Test article intake for F1-animals was indicated in the table 51. Lower accuracies were measured for the prepared powder diets. In order to determine the
worst case scenario exposure, the table 52 presents the mean of means test item intake corrected for the mean of means accuracy per period. See below tables 51 and 52 in the field 'Any other information on materials and methods incl. tables'.

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 - GENERATION FROM WEANING ONWARDS (Cohort 1A):
Hematology:
Test item-related changes were observed in males and females from 300 ppm onwards.
The mean lymphocyte count (LYMPH) was increased when compared with concurrent control for all treatment groups and both sexes (statistically significant in males at 3125 ppm and in females at 1000 and 3120 ppm), resulting in increased mean total white blood cell (WBC) counts (statistically significant only in females at 1000 and 3125 ppm). Additionally, an increase in mean large unstained cells (LUC) count was observed in all treatment groups and both sexes (statistically significant only in males at 3125 ppm). LUC are normally either larger monocytes or lymphocytes that could not be classified, and given the increase in lymphocytes that was observed in these animals, these increased LUC counts were considered test item-related as well. The fold changes for these parameters when compared with control are presented in table 53, see table 53 in the field 'Any other information on materials an methods incl. tables.

The increased mean basophil concentration for females at 300, 1000 and 3125 ppm was considered to have arisen as a result of slightly low control value
when compared with the historical control range and therefore considered to be of no toxicological significance.
Any other changes in hematology parameters were considered to be unrelated to administration of the test item due to the minimal magnitude of the change,
variation in direction of change, absence of a dose response, relatively high values in single animals and/or absence of biological relevance.

See also table 54 in the field below 'Attached backgrowd material'.

Historical Control Data for Wistar Han rats; F1-females (2017-2021): Basophils (109/L) mean = 0.0; P5 – P95 = 0.00 – 0.01 (n=114).


Coagulation:
Coagulation parameters of treated rats were considered not to have been affected by treatment. See table 55 in the field below
'Attached backgrowd material'.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

F1 - OFFSPRINGS (F1-PUPS - DURING LACTATION):
Clinical Biochemistry (T4 levels) on PND 4 F1-Pups:
Serum T4 levels in male and female pups culled at PND 4 were considered not to be affected by treatment with the test item. It should be noted that for
several animals within each group, values of 5.0 ng/mL were reported (i.e. ½ LOQ) as the original value was below LOQ, which is represented by relatively low mean values. If the 5.0 ng/mL values would not be taken into account, mean values would be 12.6, 12.9, 12.9 en 11.9 ng/mL for control, 300, 1000 and 3125 ppm groups, respectively.

See table 43 in the field below 'Attached backgrowd material'.
Note to table: Serum T4 levels of PND 4 pups are reported as females, but these were derived from pooled samples collected from male and female pups.

F1 - GENERATION FROM WEANING ONWARDS (Cohorts 1A and Cohort Surplus)
Clinical chemistry:
The following changes distinguished treated Cohort 1A animals from control animals:
• Mean concentration of total bilirubin was decreased for males at 1000 and 3125 ppm (0.89 and 0.85x of control, respectively; not statistically
significant).
Given the magnitude and based on a dose-related response, this change was considered to be test item-related.
Any other changes in clinical chemistry parameters were considered to be unrelated to administration of the test item due to the minimal magnitude of the
change, variation in direction of change, absence of a dose response, relatively high values in single animals and/or absence of biological relevance.

See table 56 in the field below 'Attached backgrowd material'.

Thyroid hormone analyses:
Cohort 1A (PND 89-95):
Serum levels of T4 and TSH in Cohort 1A males and females were considered unaffected by treatment with the test item up to 3125 ppm.
The increased mean TSH concentration in males at 3125 ppm (1.82x of control; not statistically significant) was attributed to the relatively high value in a
single animal (No. 424). As remaining individual values generally remained within concurrent control range, no relationship with the test item was indicated.
The decreased mean TSH concentration in females at 300 and 1000 ppm (0.42 and 0.61x of control, respectively; not statistically significant at 1000 ppm)
was considered unrelated to the test item in absence of a dose-related response.

Cohort Surplus (PND 22-24):
Decreased mean TSH levels were noted for PND 22-24 males and females at 3125 ppm (0.63 and 0.86x of control, respectively; not statistically significant).
Individual values generally remained within the concurrent and historical control range .
Serum T4 levels in male and female pups of Cohort Surplus at PND 22-24 were considered not to be affected by treatment with the test item.

See table 56bis in the field below 'Attached backgrowd material'.

Historical control data for TSH (mU/L) in PND 22-24 pups (2017-2021): Males mean = 0.081, P5 P95 = 0.0245 0.2065 (n=100) / Females mean = 0.076, P5-P95 = 0.0245-0.1765 (n=100).

Urinalysis findings:

no effects observed

Description (incidence and severity):

F1 - GENERATION FROM WEANING ONWARS (Cohort 1A):

Urinalysis parameters of treated rats were considered not to be affected by treatment with the test item.

See table 57 in the field below 'Attached backgrowd material'.

Sexual maturation:

no effects observed

Description (incidence and severity):

F1- Generation from weaning onwards (Cohorts 1A, 1B, 1C and Surplus):
Sexual maturation was considered not to be affected by treatment with the test item.
In all treatment groups, females were slightly older and mean body weight was slightly higher than in concurrent control females on the day that vaginal patency was reached (not statistically significant for body weight at 1000 pm). Additionally, females of the treatment groups were slightly older on the day of first estrus
than concurrent control females (not statistically significant). These differences were attributed to relatively low control values and were therefore considered
unrelated to treatment with the test item.

Historical Control Data for Wistar Han rats; F1-females (2017-2021):
- Vaginal opening (VO; PND): mean = 31.4; P5-P95 = 27.00-35.00 (n=722)
- Body weight on day of VO (gram): mean = 95; P5-P95 = 72.5-115.0 (n=720)
- First estrus (PND): mean = 35.3; P5-P95 = 31.00-39.00 (n=239)

See table 58 in the field below 'Attached backgrowd material'.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

F1 - GENERATION - F1-PUPS:
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.

See table 44 in the field below 'Attached backgrowd material'.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

F1 - GENERATION – F1-PUPS:
Treatment up to 3125 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

See table 44 in the field below 'Attached backgrowd material'.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 - GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B and Cohort Surplus):

Cohort 1A (PND 89-95)
The differences noted in males for the absolute weights of pituitary gland (at 1000 and 3125 ppm: 0.87 and 0.89x of control, respectively) and thymus (at 1000 and 3125 ppm: 0.84 and 0.86x of control, respectively), and relative weights of brain (at 3125 ppm: 1.08x of control), heart (at 3125 ppm: 1.05x of control),
liver (at 3125ppm: 1.05x of control) and kidney (at 1000 and 3125 ppm: 1.07 and 1.15x of control, respectively) were attributed to the lower terminal body
weights compared to concurrent control (statistically significant in F1 males at 1000 ppm (8% lower) and at 3125 ppm (9% lower)).
In females at 3125 ppm, there were statistically significant organ weight changes in the relative weight of the liver (1.08x of control) and spleen (1.14x control), which were considered to be mainly caused by a lower final body weight (5% lower).
Any other differences in organ weights of males and females including those that reached statistical significance (Males: absolute weights of brain and heart at 1000 ppm, prostate gland at 300 ppm, and relative weight of kidney at 300 ppm) were considered not to be 4 tert-butylpyrocatechol-related due to the lack of a dose-related pattern, lack of a test item-related microscopic correlate and/or general overlap and variability in individual values.

Cohort 1B (≥ PND 90)
There were no test item-related alterations in organ weights up to 3125 ppm.

Cohort Surplus (PND 22-24)
Weights of brain, thymus and spleen (absolute and relative to body weight) of treated PND 22-24 males and females were considered to be similar to those of control animals.


See tables 59 and 59 bis in the field below 'Attached backgrowd material'.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1-GENERATION: Macroscopy until Weaning – F1-Pups:
No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment with the test
item. For the pup of Litter No. 113 (control) that was found dead on PND 18, the stomach was noted to be grown together with the pancreas at necropsy.
Additionally, for the majority of the pup(s) of Female Nos. 125 (control), 134 (300 ppm), 164, 171, 173 (1000 ppm) that were found dead at first litter check,
beginning autolysis and/or absence of milk in the stomach were noted. For some pups cannibalism was observed. The nature and incidence of these and
other macroscopic findings remained within the range considered normal for pups of this age or occurred in the control group only, and were therefore
considered unrelated to the test item.

See table 45 in the field below 'Attached backgrowd material'.

F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Suplus)

Cohort 1A (PND 89-95)
There were no test item-related macroscopic observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Cohort 1B (≥ PND 90)
There were no test item-related macroscopic observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Cohort 1C (males: ≥ PND 35; females: ≥ PND 25)
There were no test item-related macroscopic observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Cohort Surplus (PND 22-24)
No macroscopic abnormalities were observed.


See table 60 in the field below 'Attached backgrowd material'.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Histopathology – PND 4 F1-PUPS:
There were no test item-related microscopic findings in the stomach of the selected PND 4 pups.
The only recorded finding was a cyst in the stomach of a control male pup.

See table 46 in the field below 'Attached backgrowd material'.


F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A and Suplus):

Cohort 1A (PND 89-95):
Test item-related microscopic findings after treatment with 4-tert-butylpyrocatechol were noted in the stomach of the 3125 ppm males and females and are
summarized in the table 59. See table 59 in the field below 'Any other information on results incl. tables'.
Non-glandular stomach (i.e. forestomach): Hyperkeratosis was present at a high incidence and up to slight degree in both sexes at 3125 ppm. In some
animals, this was accompanied by diffuse squamous cell hyperplasia (up to slight degree) and or focal erosions of the non glandular stomach. The minimal
diffuse hyperkeratosis in a 1000 ppm male and female were considered to be spontaneous findings.
A finding of note was present in a single 3125 ppm male (No. 424) consisting of marked testicular tubular atrophy (mostly Sertoli cell only) and a massive
reduction of the presence of sperm in the lumen of the epididymides, correlating in both organs with the macroscopic finding reduced size. This was
considered to be unrelated to the test item due to the single incidence and absence of test item-related findings in the testes or epididymides and presence of normal progression of the spermatogenic cycle or the expected cell associations and proportions in the various stages of spermatogenesis in all other males of Cohort 1A.
A follicular cell adenoma, a benign tumor, was detected in the thyroid gland (unilateral) of a female at 3125 ppm (No. 705). Since there was no further
evidence of test item-related proliferative lesions in the thyroid gland and this tumor occurred in a single animal, this was regarded as a spontaneous finding.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

There were no test item-related effects on the ovarian follicle and corpora lutea counts in the F1 3125 ppm group females (Cohort 1A) when compared to control group females. Any variations between group mean counts represented biological variability and were not statistically significant.

See table 61 in the field below 'Any other information on results incl. table'.

Cohort Surplus (PND 22-24):
There were no test item-related findings in the stomach. The few microscopic findings noted (inflammation in a single control male and dilated glands in a few animals) did not distinguish treated animals from control animals and were regarded to be spontaneous in nature.

See table 46 in the field below 'Attached backgrowd material'.

Other effects:

no effects observed

Description (incidence and severity):

ESTROUS CYCLE - F1-GENERATION FROM WEANING ONWARDS (Cohort 1A):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had a regular cycle of 4-5 days between
PND 75 to 88. For one female at 1000 ppm (No. 624) the cycle classification could not be determined and one female at 3125 ppm (No. 710) was acyclic.
Given the incidental nature, these findings did not indicate a relation with the test item.
See table 62 in the field below 'Attached backgrowd material'.

SPLENIC LYMPHOCYTE SUBPOPULATION - F1-GENERATION FROM WEANING ONWARDS (Cohort 1A):
There were no test item-related changed in splenic lymphocyte subpopulations of Cohort 1A animals.
A statistically significantly higher NK-cell subpopulation of splenic lymphocytes was observed for Cohort 1A females at 3125 ppm (1.25x of control). This shift was considered to represent biological variability and therefore not to be related to treatment with the test item, because this shift was slight of nature and
occurred in the absence of either a dose-related trend or apparent changes in lymphoid cellularity of the spleen as revealed by histopathology evaluation.
There were no indications that other splenic lymphocyte subpopulations were affected in Cohort 1A males or females.
See table 63 in the field below 'Attached backgrowd material'.

SPERM ANALYSIS - F1-GENERATION FROM WEANING ONWARDS - Cohort 1A):
There were no test item-related changes in sperm motility, concentration and morphology in males treated up to 3125 ppm.
See table 64 in the field below 'Attached backgrowd material'.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 3 125 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect (general toxicity)

Remarks on result:

other:

Remarks:

No adverse effects were observed.

2

Dose descriptor:

LOEL

Generation:

F1

Effect level:

ca. 300 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

clinical biochemistry

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

1) Body weight: Mean body weight gain was reduced in males at 1000 and 3125 ppm, resulting in a lower mean body weight throughout the treatment period. Mean terminal body weights of Cohort 1A and 1B animals were 8 and 1% lower than control for males at 1000 ppm, and 9 and 6 lower than control for males at 3125 ppm, respectively.As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse. 2) Hematology and clinical biochemistry: In F1-generation animals at 300, 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with adverse anatomic pathology and as such they were regarded as non-adverse. 3) Effect on the glandular stomach: at 3125 ppm of TBC diffuse hyperkeratosis (up to slight degree) with or without squamous cell hyperplasia (Cohort 1A) were observed. Since the recorded severities of these findings remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, these findings was considered as a local, non-adverse test item-related effect.

3

Dose descriptor:

NOEL

Generation:

F1

Effect level:

< 300 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

clinical biochemistry

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

1) Body weight: Mean body weight gain was reduced in males at 1000 and 3125 ppm, resulting in a lower mean body weight throughout the treatment period. Mean terminal body weights of Cohort 1A and 1B animals were 8 and 1% lower than control for males at 1000 ppm, and 9 and 6% lower than control for males at 3125 ppm, respectively. As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse. 2) Hematology and clinical biochemistry: In F1-generation animals at 300, 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with adverse anatomic pathology and as such they were regarded as non-adverse. 3) Effect on the glandular stomach: at 3125 ppm of TBC diffuse hyperkeratosis (up to slight degree) with or without squamous cell hyperplasia (Cohort 1A) were observed. Since the recorded severities of these findings remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, these findings was considered as a local, non-adverse test item-related effect.

4

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 3 125 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental effect.

Remarks on result:

other:

Remarks:

Developmental toxicity: No developmental toxicity was observed up to the highest dose level tested (3125 ppm).

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

DIET ANALYSES:

Accuracy

In the chromatograms of the Group 1 diets prepared for use in Weeks 3 and 26, a small response was observed at the retention time of the test item. It was considered not to derive from the diet since a similar response was obtained in the analytical blanks. The maximum contribution to the Group 2 samples was 0.71% based on peak area and taking dilution factors into account. In the diets of Group 1 prepared for use in Weeks 1, 6, 11, 14, 15, 16 and 21, no test item was detected.

An overview of the obtained accuracy results for Group 2, 3 and 4 diets is given in the tables below.

Table 19: Mean Accuracy of Diets F0-Generation from Premating up to Lactation

Occasion	Study Phase	Group 2 (300 ppm)	Group 3 (1000 ppm)	Group 4 (3125 ppm)
Week 1 of treatment	Premating period	67%	69%	78%
Week 3 of treatment		61%	67%	75%
Week 6 of treatment		68%	76%	82%
Mean of Means Accuracy Premating Period		65 %	71%	78%
Week 11 of treatment	Mating period	76%	80%	83%

 

Table 20: Mean Accuracy of Diets F0-Generation during Lactation

Occasion	Study Phase	Group 2^a	Group 3^a	Group 4^a
Week 14 of treatment	Days 1-3	70%	73%	77%
Week 15 of treatment	Days 4-6	69%	68%	73%
Week 16 of treatment	Day 7 onwards	63%	70%	72%

^a  During the lactation period, the following dietary concentrations were used (based on historical control data for relative food consumption):

Lactation	Group 2	Group 3	Group 4
Days 1-3	200 ppm	666 ppm	2085 ppm
Days 4-6	150 ppm	500 ppm	1563 ppm
Day 7 onwards	120 ppm	400 ppm	1250 ppm

 

Table 21: Mean Accuracy of Diets F1-Generation

Occasion	Group 2 (300 ppm)	Group 3 (1000 ppm)	Group 4 (3125 ppm)
Week 21 of treatment	70%	73%	81%
Week 26 of treatment	75%	78%	81%
Mean of Means Accuracy Treatment Period	73%	76%	81%

For the diet samples of Groups 2, 3 and 4, prepared for use in Weeks 1, 3, 6, 11, 14, 15, 16, 21 and 26, the mean accuracy of the diet samples was 61 – 83%. Mean accuracy seemed to increase with an increase in the target concentration of the diet. Since the mean accuracy was not always within the acceptance criterion of 80 – 120 %, several out of specification investigations were performed. During these investigations, the diet preparation procedure as well as the analytical procedure was scrutinized. As part of the investigation for samples from diets prepared for Week 3 of treatment, presence of test item – metal complexes or oxidized test item (Quinones) which may absorb light at a different wavelength was checked in the extracts. The possibility of oxidation was based on information provided by the sponsor and published literature. UV-Vis spectra from 200-900 nm were recorded for one sample of each group. The spectra were recorded after extraction, filtration and further dilution if required. These spectra were compared with spectra for blank extraction solvent and with spectra for two calibration solutions. Since it was not possible to draw a conclusion from the UV-Vis spectra, the samples were subsequently analyzed on the UPLC-UV using the validated method, with UV detection at a general wavelength of 210 nm. The chromatograms of the calibration solutions and diet samples showed one major test item peak at 1.4 min. In the spectra of the diet samples, there were several peaks visible between 1.8 and 4.0 min which were attributed to the diet matrix. No other test item-related peaks such as test item - metal complexes or oxidized test item (Quinones) were visible. However, no detailed examination or identification of these peaks has been carried out, thus it is not possible to assert that these peaks were linked only to the diet matrix.

The conclusion of all out of specification investigations was that no explanation could be found for the low mean accuracy results for the diet samples of Groups 2, 3 and 4.

Homogeneity

The diets of Groups 2 and 4 were homogeneous (i.e. coefficient of variation≤ 10%).

F0 - GENERATION RESULTS:

TEST ITEM INTAKE:

Table 26: Test article intake over the study period (F0 - generation).

 

		Mean of Means Intake [mg Test Item/kg Body Weight] (Mean Range Indicated within Brackets)
	Group No.	2		3		4
	Nominal Dietary Inclusion Level (ppm)^(b)	300		1000		3125
Sex	Study Period
Males	Pre-mating	21	(16-30)	70	(55-98)	221	(177-303)
	Post-mating	15	(13-16)	52	(48-56)	173	(159-195)
	Mean of Means^(a)	20		66		211
Females	Pre-mating	24	(20-31)	82	(68-103)	249	(210-310)
	Post-coitum Days 0-23 Post-coitum Days 0-24	- 22	- (12-26)	74 -	(41-85) -	218 -	(151-255) -
	Lactation Days 1-3	22	(18-26)	80	(65-94)	251	(207-276)
	Lactation Days 4-6	21	(21-21)	75	(72-79)	241	(230-247)
	Lactation Days 7-24	23	(15-31)	80	(43-102)	254	(134-334)
	Mean of Means ^(a)	23		80		243

- = not applicable.

(a) Mean of means of all periods, weighed for number of measurement intervals per period: Males ((70 * mean premating) + (18 * mean mating)) / 88, Females: ((70 * mean premating) + (23 or 24 mean post-coitum) + (23 * mean lactation)) / 116 or 117.

(b) During the lactation period, dietary concentration of 4 -tert-butylpyrocatechol were lowered to maintain constant test item (based on historical control data for relative food consumption).

Table 27: Corrected test article intake over the study period (F0 -generation).

		Mean of Means Intake[mg Test Item/kg Body Weight] Corrected for Mean of Mean Accuracy of Prepared Diets
	Group No.	2	3	4
	Nominal Dietary Inclusion Level (ppm)	300	1000	3125
Sex	Study Period
Males	Pre-mating	14	50	172
	Post-mating	11	42	144
Females	Pre-mating	16	58	194
	Post-coitum Days 0-23 Post-coitum Days 0-24	- 17	59 -	181 -
	Lactation Days 1-3	16	59	193
	Lactation Days 4-6	14	51	176
	Lactation Days 7-24	14	56	183

 

HISTOPATHOLOGY:

Table 35: Summary of test item-related microscopic findings (F0 - generation).

 

	Males				Females
Dose level (ppm):	0	300	1000	3125	0	300	1000	3125
NON-GLANDULAR STOMACH^a	25	25	25	25	25	25	25	25
Squamous cell hyperplasia
Minimal	-	-	-	5	-	-	-	2
Hyperkeratosis, diffuse
Minimal	-	1	1	22	1	3	2	21
Slight	-	-	-	3	-	-	-	-

^{a = Number of tissues examined from each group.}

F1- GENERATION:

- F1 - GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Surplus):

TEST ITEM INTAKE:

Table 51: Mean test article intake (F1 - generation)

		Mean of Means Intake [mg Test Item/kg Body Weight] (Mean Range Indicated within Brackets)
	Group No.	2		3		4
	Nominal Dietary Inclusion Level (ppm)	300		1000		3125
Sex	Study period
Males	Treatment	26	(15-42)	85	(48 - 139)	273	(175-439)
Females	Treatment	27	(20-41)	89	(65-134)	284	(217-436)

 

Table 52: Corrected mean test article intake (F1 generation):

		Mean over Means Intake [mg Test Item/kg Body Weight] Corrected for Mean of Mean Accuracy of Prepared Diets
	Group No.	2	3	4
	Nominal Dietary Inclusion Level (ppm)	300	1000	3125
Sex	Study period
Males	Treatment	19	65	221
Females	Treatment	20	68	230

F1 -GENERATION - COHORT 1A

Table 53: Summary Test Item-Related Hematology Changes (fold change to Control) – F₁-Cohort 1A

 

	Males			Females
Dose level (ppm):	300	1000	3125	300	1000	3125
No. of animals analyzed	10	10	10	10	10	10
WBC (10^9/L)	1.19	1.20	1.34	1.15	1.39*	1.39*
LYMPH (10^9/L)	1.26	1.25	1.49**	1.17	1.41*	1.47**
LUC (10^9/L)	1.11	1.41	2.14^#	1.46	1.62	1.92

 

^#Kruskal-Wallis & Dunn: = p ≤ 0.05

* / ** Anova & Dunnett: * = p ≤ 0.05, ** = p ≤ 0.01

Table 61. Summary test-item-related microscopic findings - F1 -Cohort 1A.

 

	Males				Females
Dose level (ppm):	0	300	1000	3125	0	300	1000	3125
NON-GLANDULAR STOMACH^a	20	20	20	20	20	20	20	20
Erosion,
Minimal	-	-	-	2	-	-	-	2
Squamous cell hyperplasia
Minimal	-	-	-	7	-	-	-	8
Slight	-	-	-	3	-	-	-	4
Hyperkeratosis, diffuse
Minimal	-	-	1	13	-	-	1	11
Slight	-	-	-	5	-	-	-	8
^a = Number of tissues examined from each group.

 

Applicant's summary and conclusion

Conclusions:

- This extended one generation showed that TBC induced mean body weight gain reduction in males and females F0-generation animals in presence of TBC at
3125 ppm, resulting in a lower mean body weight during the pre-mating and mating period and in males F1-generation males at 1000 and 3125 ppm, resulting in a
lower mean body weight throughout the treatment period.
- At 3125 ppm (the higher tested dose), test item-related findings were present in the non-glandular stomach (forestomach) in adult rats. These test item related findings consisted of an increased incidence and severity of diffuse hyperkeratosis with or without squamous cell hyperplasia. Similar effects were observed to the F1-generation animal non-glandular stomach at 3125 ppm. The glandular stomach of F1-rats of Cohort 1A showed a higher incidence and severity of squamous cell hyperplasia compared to the stomachs of the F0-generation at 3125 ppm.

Based on this study, no general, reproductive or developmental toxicity was observed up to the highest dose tested (3125 ppm).

Executive summary:

The objective of this study was to provide an evaluation of the pre- and postnatal effects of 4-tert-butylpyrocatechol on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of 4-tert-butylpyrocatechol on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation. The dose levels in this study were selected to be 0, 300, 1000 and 3125 ppm, based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with dietary exposure of 4-tert-butylpyrocatechol in rats. The same dietary concentrations were used throughout the study, except for the lactation period. As food intake is considerably higher in lactating females, dietary concentrations were lowered for all treated groups (based on historical control data for relative food consumption) during the lactation period (PND 1-24). Chemical analyses of dietary preparations were conducted at nine occasions during the study to assess accuracy and homogeneity.

For the F0-generation, the following parameters and endpoints were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. For the F1-generation, the following parameters and endpoints were evaluated: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, estrous cycle, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined for the F0-generation: mating and fertility indices, precoital time, estrous cycle determination, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones) and histopathological examination of the stomach (PND 4 and PND 22-24 pups).

Results: Analysis of diet preparations confirmed that the test item was homogeneously distributed in the diet. The achieved dietary concentrations of 4-tert-butylpyrocatechol were however not always within the acceptance criterion of 80 – 120 %. A thorough check of the diet preparation procedures and analytical methodology revealed no explanation for the lower accuracies. In order to determine the worst case scenario exposure, test item intake was corrected for the mean achieved dietary concentrations per study period.

No mortality occurred during the study period that was considered to be related to treatment with the test item. In the F0-generation, one female of the control and 1000 ppm groups each, were euthanized on Lactation Day 2 or 1, respectively, as these females had a total litter loss. In the F1-generation, one male (1000 ppm, Cohort 1A) was sacrificed 5 days post weaning as it was assigned to the females. No general, reproductive, or developmental toxicity was observed up to the highest dose tested (3125 ppm).

The following test item-related, non-adverse, effects were observed:

Parental results: Mean body weight gain was reduced in males and females at 3125 ppm, resulting in a lower mean body weight during the pre-mating and mating period. For females at 3125 ppm, no relevant differences were noted during the gestation period, whereas a higher mean body weight gain was observed from Day 7 of lactation onwards resulting in a slight recovery in mean body weight. Mean terminal body weight was 9 and 4% lower than concurrent control mean for males and females, respectively. As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse.

In animals at 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected by treatment with the test item, including decreased platelets counts in females, and decreased mean bile acid and creatinine concentrations in males. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology and as such they were regarded as non-adverse. In addition, mean serum levels oft hyroid stimulating hormone (TSH) were dose-dependently increased for females at all dose levels. As individual values generally remained within the historical control range, the change in serum TSH in the females was considered not to be adverse. 

At 3125 ppm, test item-related findings were present in the non-glandular stomach (forestomach) in adult rats. These test item-related findings consisted of an increased incidence and severity of diffuse hyperkeratosis (up to slight degree) with or without squamous cell hyperplasia. Since the recorded severities of these findings in adults remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, the combination of these findings was considered as a local, non-adverse test item-related effect.

Developmental results: Mean serum levels of TSH were decreased for male and female PND 22-24 pups (Cohort Surplus) at 3125 ppm. As individual values remained within the historical control range, the change in serum TSH was considered not to be adverse. 

F1-generation results: Mean body weight gain was reduced in males at 1000 and 3125 ppm, resulting in a lower mean body weight throughout the treatment period. Mean terminal body weights of Cohort 1A and 1B animals were 8 and 1% lower than control for males at 1000 ppm, and 9 and 6% lower than control for males at 3125 ppm, respectively. As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse.

In animals at 300, 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected, including increased mean lymphocyte counts and as a result increased mean total white blood cell count and mean large unstained cell count in both sexes, and decreased mean total bilirubin concentration in males. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with adverse anatomic pathology and as such they were regarded as non-adverse.

Similar to the F0-generation, test item-related findings were present in the non-glandular stomach at 3125 ppm. These test item‑related findings consisted of an increased incidence and severity of diffuse hyperkeratosis(up to slight degree) with or withoutsquamous cell hyperplasia.The glandular stomach of F1-rats of Cohort 1A showed a higher incidence and severity of squamous cell hyperplasia (incidence of 50% in males and females 60%, up to slight degree) compared to the stomachs of the F0-generation at 3125 ppm (incidence of 20% in males and 8% in females, at minimal degree). Furthermore, there were focal erosions (minimal degree) in the non-glandular stomach in 2/20 F1-rats of both sexes at 3125 ppm. Since the recorded severities of these findings remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, the combination of these findings was considered as a local, non-adverse test item-related effect.

In conclusion, based on the results of this extended one-generation reproductive toxicity study (including Cohorts 1), the following No Observed Adverse Effect Levels (NOAELs) of 4-tert-butylpyrocatechol were established:

General toxicity NOAEL:     

F0-generation : at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 172-194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).
F1-generation: at least 3125 ppm (on average corresponding to a corrected test article intake of 221 and 230 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).

Reproduction NOAEL: at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 176-194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).

Developmental NOAEL: at least 3125 ppm (on average corresponding to a corrected test article intake of 144 -172 and 176 -194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).

The test item (4 -tert-butylpyrocatechol) did not induce reprotoxic effects neither developmental effects up to the highest tested dose (3125 ppm)