5-amino-o-cresol

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Apr. 22, 2002 to Aug. 28, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

According to OECD principles of GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-o-cresol

EC Number:

220-618-6

EC Name:

5-amino-o-cresol

Cas Number:

2835-95-2

Molecular formula:

C7H9NO

IUPAC Name:

5-amino-2-methylphenol

Constituent 2

Reference substance name:

4-amino-2-hydroxytoluene

IUPAC Name:

4-amino-2-hydroxytoluene

Constituent 3

Reference substance name:

Haarpurpur

IUPAC Name:

Haarpurpur

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: 4-amino-2-hydroxytoluene
- TSIN: 23032
- Substance type: Pure active substance
- Physical state: Brownish crystalline powder
- Stability under test conditions: Stable under storage conditions employed. Test substance was stable for at least 2h in the vehicle.
- Storage condition of test material: At room temperature in the dark
- Solubility: 8g/L in water (20°C)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar Crl:(WI) BR (outbred, SPF-Quality) was obtained from Charles River Deutschland (Sulzfeld, Germany)
- Age at study initiation: 5-6 weeks (F0 males); 13-14 weeks (F0 females)
- Weight at study initiation: 173-210 g (F0 males); 215-268 g (F0 females)
- Housing: Upon arrival, F0 animals were housed in groups of 4 animals/sex/cage in suspended stainless steel cages. Mated females and males were individually housed in labeled polycarbonate cages containing sawdust as bedding material.
- Diet: Standard pelleted laboratory animal diet (from Altromin); ad libitum.
- Water: Tap water; ad libitum
- Analysis of bedding, diet and water did not reveal any findings that were considered to have affected the study integrity.
- Acclimation period: At least 5 days prior to initiation of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 18-24 °C
- Humidity: 30-70 %
- Incidental deviation from maximum level of relative humidity (max. 97%) and temperature (max. 26°C) did occur. Based on laboratory data this was considered not to have affected the animals.
- Air changes: Not reported
- Photoperiod: 12 hour light/12 hour dark cycle per day

IN-LIFE DATES: From: Apr. 22, 2002 To: Aug. 28, 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance was dissolved in vehicle and homogenized to a visually acceptable level.
- Rate of preparation of dosing solutions: Daily within 4 hours prior to dosing
- Storage temperature of dosing solutions: Ambient temperature

TEST MATERIAL
- Amount(s) applied: 5 mL/kg bw for first 9 days of treatment. Thereafter (from May. 01, 2002 onwards) 8 mL/kg bw for all groups, as the highest dose formulation was difficult to dose. Actual dose volumes were calculated according to the latest body weight.
- Concentration: 0, 8, 40 and 200 mg/mL initially; From May. 01, 2002 onwards: 0, 5, 25 and 125 mg/mL
- Constant concentration used: No

VEHICLE
- Justification for use and choice of vehicle: Based on information provided by the sponsor
- Amount of vehicle: 5 mL/kg bw for first 9 days of treatment. Thereafter (from May. 01, 2002 onwards) 8 mL/kg bw for all groups. Actual dosing volumes were calculated according to the latest body weight.
- Lot/batch no: Not reported
- Purity: Not reported

Details on mating procedure:

- M/F ratio per cage: 1M/1F; Females were paired on a one-to-one-basis with males from the same treatment group.

- Proof of pregnancy: Each morning following pairing, the trays under the cages were checked for ejected copulation plugs. The day on which a copulation plug was found was designated Day 0 of gestation (= day 0 post-coitum)

- After 10 days of unsuccessful pairing male partner was replaced by another proven male..

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of accuracy, homogeneity and stability were done according to a validated method. Analyses of test formulations were performed at weeks 1, 8 and 15 for all dose groups and at week 2 for groups 2 and 4 only.

- Analysis of the formulations prepared on at weeks 1, 8 and 15 revealed values for accuracy within the range of 90-97%, 98-109%, and 85-112%, respectively.

- Analysis of homogeneity of low and high dose formulations prepared on at weeks 1, 8 and 15 showed values between 95-102%, 96-107%, and 94-107%, respectively. These results were considered to represent an acceptable level for formulations of this type.

- Stability analysis of the low and high dose formulations prepared on week 1 showed a decrease over 4 hours of 3% for both dose groups, and over 7 days of 2% for both dose groups, and were thus considered stable for up to 7 days at room temperature.

Duration of treatment / exposure:

- Males: 10 weeks prior to mating up to sacrifice (the mean duration of treatment of males surviving to planned necropsy was 98 days, with a minimum of 94 days and a maximum of 102 days)

- Females: 2 weeks prior to mating up to sacrifice (the mean duration of treatment of females surviving to planned necropsy was 59 days, with a minimum of 46 days and a maximum of 72 days).

Frequency of treatment:

Once daily at approximately the same time each day

Details on study schedule:

- Age at mating of the mated F0 animals in the study: 15-16 weeks (both males and females)

- The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). Females that were littering were left undisturbed.

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle control), 40, 200 and 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

24 animals/sex/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on two 90-day toxicity studies in rats by gavage.

- In the first study (at dose levels of 300, 900 and 2700 mg/kg bw/day) animals of the 900 mg/kg bw/day dose group showed clinical symptoms, increased kidneys and liver weights, and histopathological findings. Animals of the 300 mg/kg bw/day dose group showed clinical symptoms and increased liver weights.
In the second study (at dose levels of 20, 60 and 180 mg/kg bw/day); no treatment related findings were noted up to 180 mg/kg bw/day.

- The highest dose level, 1000 mg/kg bw/day was chosen, as no severe effects were expected as determined in the first 90-day study. In addition, this level is the maximum that should be tested in a reproductive study according to the guideline.

- Rationale for animal assignment: Animals were randomized to different experimental groups at least 5 days before study start, by computer-generated random algorithm according to body weight, with all animals falling within ± 20% of the sex mean weight.

Animals were divided into following experimental groups
- Group 1: 0 mg/kg b.w./day (vehicle control)
- Group 2: 40 mg/kg b.w./day (low dose group)
- Group 3: 200 mg/kg b.w./day (mid dose group)
- Group 4: 1000 mg/kg b.w./day (high dose group)

- REPRODUCTION PROCESSES: Mating date, identity of male partner, confirmation of pregnancy, and delivery day was recorded for each female.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations included: Mortality/viability assessment; animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons. The time of death was recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS: Yes; The time of onset, degree and duration of the symptoms were recorded. Cage debris of pregnant females was examined to detect abortion or premature birth. Signs of difficult or prolonged parturition were recorded.
- Time schedule: Once daily
- How many animals: All

BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of exposure and weekly thereafter until necropsy. Mated females were weighed on gestation Day 0, 7, 14 and 21 and during lactation on Days 1, 4, 7, 14 and 21.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly for males and females (during the mating period analysis of food consumption was suspended). Food consumption of mated females was measured on gestation Days 0, 7, 14 and 21, and during lactation on Days 1, 4,7,14 and 21 post partum.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Relative food consumption for each animal determined and mean daily diet consumption calculated as g food/kg bw/day: Yes

WATER CONSUMPTION: No; Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

LACTATION: Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, were recorded.

Oestrous cyclicity (parental animals):

Not reported

Sperm parameters (parental animals):

Parameters examined in male parental generations: Testis, epididymides and seminal vesicles (with coagulating gland and fluids) were weighed for all males in parental generations.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes: The size of each litter was adjusted by eliminating extra pups to yield, as nearly as possible, 4 pups/sex/litter. Whenever the number of male or female pups prevented having 4/sex/litter, partial adjustment (for example, five males and three females) was done. Adjustments were not applied to litters of less than eight pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- The numbers of live and dead pups: Yes; first litter check (at Day 1 of lactation) and daily thereafter
- The weight of all live pups: Yes; recorded at Day 1, 4, 7, 14 and 21 of lactation
- Sex of all pups (by assessment of the ano-congenital distance): Yes
- The number of pups with physical or behavioural abnormalities daily: Yes

GROSS EXAMINATION OF DEAD PUPS: Yes
- Offspring found dead or killed before Day 14 of lactation was sexed and externally examined if practically possible. The stomach was examined for the presence of milk.
- Offspring found dead or killed on or after Day 14 of lactation was sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs. Descriptions of all macroscopic abnormalities were recorded.
- If possible, defects or cause of death were evaluated.
- No pups were preserved.

Postmortem examinations (parental animals):

SACRIFICE: All animals surviving to the end of the observation period and all moribund animals were anaesthetized using iso-flurane and subsequently exsanguinated.
- Males were killed as soon as possible after successful delivery of the dams.
- Females were killed on Day 21 post partum or shortly thereafter.

GROSS NECROPSY:
- After sacrifice or death all parental animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- In case a female was not pregnant, the uterus was stained using the Salewski technique in order to determine any very early post-implantation losses (=implantation site scars).

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organ Weights: The weights of following organ (and terminal body weight) were recorded from the surviving parental animals on the scheduled day of necropsy: Cervix with uterus, epididymides (total weight for both), kidney, liver, ovaries, pituitary gland, prostate gland, seminal vesicles with coagulating gland and fluids, testes.

- Histopathology: Kidney and liver from all animals of all groups were processed, embedded and cut at a thickness of 2-4 microns and stained with hematoxylin and eosin and examined by the pathologist. All abnormalities were recorded.

Postmortem examinations (offspring):

SACRIFICE

- All remaining pups (left after Day 4 adjustment (culling)) were sacrificed on Day 21 post partum or shortly thereafter by carbon dioxide asphyxiation.

GROSS NECROPSY
- Pups found dead or killed before Day 14 of lactation was sexed and externally examined if practically possible. The stomach was examined for the presence of milk.

- Pups found dead or killed on or after Day 14 of lactation was sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs. Descriptions of all macroscopic abnormalities were recorded. If possible, defects or cause of death were evaluated.

- No pups were preserved


HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.

- The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data could not be assumed to follow a normal distribution.

- The exact Fisher-test was applied for 2x2 tables if variables could be dichotomized without loss of information. All tests were two-sided and in all cases p< 0.05 was accepted as the lowest level of significance.

Reproductive indices:

Percentage mating: [Number of females mated / Number of pregnant females] x 100
Fertility index: [Number of pregnant females / Number of females paired] x 100
Conception rate: [Number of pregnant females / Number of females mated] x 100
Gestation index: [Number of females bearing live pups / Number of pregnant females] x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Viability index: [Number of live pups on day 4 post-partum / Number of pups born alive] x 100
Weaning index: [Number of live pups on day 21 post-partum / Number of pups on day 4 post-partum] x 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] x 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] x 100
Percentage of postnatal loss days 0 - 4 post partum: [Number of dead pups on day 4 post partum / Number of live pups at First Litter Check] x 100
Percentage of breeding loss day 5 until weaning: [Number of dead pups between days 5 and 21 post partum / Number of live pups on day 4 post partum] x 100
Percentage live males at weaning: [Number of live male pups on day 21 post partum / Number of live pups on day 21 post partum] x 100
Percentage live females at weaning: [Number of live female pups on day 21 post partum / Number of live pups on day 21 post partum] x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- A total of 11 deaths (2 mid dose and 9 high dose group) were recorded during the study. The deaths of 2 males and 5 females of the high dose group were considered to be treatment related.
- Treatment-related clinical signs observed in high dose group animals consisted of tonic spasms, lateral recumbency, rales, gasping, emaciation, hypothermia, lethargy, labored respiration, pale appearance, chromodacryorrhoea on the snout, and ptosis.
- All males and females of the mid and high dose groups showed brown urine during treatment. An increase in brown or yellow staining of several body parts was observed for males and females of high dose group. This discoloration was due to the staining properties of the test substance, and thus not considered toxicologically relevant.
- Brown or yellow staining of several body parts was also observed in animals of other groups, however with a much lower incidence and on fewer occasions.
- Incidental findings consisted of alopecia, scabs, hunched posture, piloerection, wound, and missing tail apex. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Body weights and body weight gain of males of the high dose group were statistically significantly decreased during the complete treatment period. Body weight gain of females of the high dose group was statistically significantly decreased on Days 4 and 7 of the lactation period.
- Body weight of females of the high dose group was statistically significantly increased on Day 1 of the mating period. As this finding was incidental, it was not considered to be toxicologically relevant.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Food consumption of males of the high dose group was statistically significantly decreased on Days 1-15, and 57-64 of the pre-mating period. Food consumption of females of the high dose group was statistically significantly decreased on Days 8-15 of the pre-mating period, and on Days 1-21 of the lactation period.
- Relative food consumption of females of the high dose group was statistically significantly decreased on Days 14-21 of the post-coitum period, and on Days 1-21 of the lactation period.
- Statistical significant increases in relative food consumption were observed during several days of the post-mating period in males of the mid and high dose groups. This finding was not considered to be an adverse effect.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Females of the high dose group showed a slightly increased mean duration of gestation, a decreased gestation index, the occurrence of delivery difficulties, and a decrease in number of pups at birth.
- In the control group, one female was non-pregnant and two females did not mate within the first mating period but in the second mating period.
- In the low dose group, one female was non-pregnant, one female showed implantation sites only, and one female did not mate within the first mating period but in the second mating period.
- In the mid dose group, one female was non-pregnant, and one female died spontaneously two days after mating.
- In the high dose group, two females were non-pregnant, and three females showed delivery difficulties.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test compound. All the findings were considered to be either incidental or consistent with historical control observation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Treatment related increased absolute and relative liver weight; and increased relative testes weights were observed in high dose group males.
- Mid and high dose group males showed statistically significantly increased relative kidneys weights. However, this finding was determined to have no relevance in human safety assessment.
- Males of the low and mid dose groups showed statistically significantly increased relative liver weights, and females of the high dose group showed statistically significantly increased relative and absolute kidneys weights. In the absence of any histopathological effects, these findings were considered not to be toxicologically relevant.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- In high dose group males, an increased incidence and severity (primarily slight or moderate) of midzonal/centrilobular hypertrophy in the liver was observed. - In high dose group females minor grades of hepatocellular vacuolation were observed. These finding was considered to be treatment related.
- In the kidneys of male rats cortical hyaline droplets were recorded at an increased incidence and severity at mid and high dose group. These droplets were considered to represent alpha2µglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules.
- A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium which may result in tubular cell degeneration. At high dose level this alteration was accompanied by an increase in severity of corticomedullary tubular atrophy and hyaline casts. Hyaline casts occurred in greater number at mid dose than in controls. The slightly increased severity of cortical hyaline droplets recorded at low dose group, was not associated with indications of tubular epithelial degeneration.
- These changes were exclusively found in male rats; therefore, these findings are not predictive for man and their occurrence has no relevance in human safety assessment.
- There were no treatment related alterations in the kidneys of female rats.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

200 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

Parental toxicity

Effect level:

200 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Increased mortality rate, severe clinical signs and reduced body weight gain and feed consumption

Results: F1 generation

General toxicity (F1)

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY: (OFFSPRING)
- The number of dead pups at first litter check and postnatal loss during Days 0-4 post-partum were increased in high dose group. Due to this, the number of living pups at first litter check, at Day 4 post-partum, and at Day 21 post-partum, and the viability index were decreased in this group.
- Breeding loss during Days 5 and 21 post-partum was statistically significantly increased and the weaning index was statistically significantly decreased in litters of the mid dose group. This was mainly due to the spontaneous death of dam 156 on day 20 post-partum, of which then all eight pups had to be sent to necropsy. These findings were thus not considered to be toxicologically significant.
CLINICAL SIGNS: (OFFSPRING)
- Several pups of the high dose group showed very bad health.
- Incidental clinical symptoms consisted of small, pale or cold appearance, discoloration of several parts of the body, little or no milk, little hair, alopecia, scabs, scales, a wound, a hemorrhage, bruises, abnormal posture of the head, emaciated, lethargy, dying.
- One pup of the mid dose group showed an absent tail, and one pup of the high dose group showed an absent right hind leg and a shortened tail. These findings were considered incidental, and were not considered to be caused by treatment.
BODY WEIGHT: (OFFSPRING)
- As compared to control, mean body weights of high dose group pups were statistically significantly decreased on Day 14 and 21 of lactation.
GROSS PATHOLOGY: (OFFSPRING)
- Macroscopic examination of the pups revealed pelvic dilation of the right kidney, pelvic dilation of both kidneys, cannibalism, enlarged spleen, testes reduced in size, epididymides reduced in size, spleen reduced in size. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

200 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Increased post-natal loss resulting in a decreased viability index, as well as an increased incidence of clinical signs and decreased body weights in the offspring.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Breeding Data (F0 Generation), (Study# 71410) Parameters Values Group 1 / control Group 2 / 40 mg/kg bw Group 3 / 200 mg/kg bw Group 4 / 1000 mg/kg bw Litters Total 23 22 22 17 Duration of gestation Mean* 21.3 21.1 21.4 22.1 Dead pups at first litter check Litters affected (#) 1 2 4 7 Total 1 3 8 38 Mean* 0.0 0.1 0.4 2.2 Living pups at first litter check % of males / females (# 53/47 52/48 52/48 44/56 Total 326 293 295 153 Mean* 14.2 13.3 13.4 9 Postnatal loss days 0-4 p.p. % of living pups 4 3.1 7.5 15.7 Litters affected (# ) 8 6 8 7 Total (# ) 13 9 22 24 Mean* 0.6 0.4 1 1.4 Culled pups Total 129 111 111 41 Living pups day 4 p.p. Total 184 173 162 88 Mean* 8 7.9 7.4 5.2 Breeding loss days 5 - 21 p.p. % of living pups at day 4 p.p. 2.2 5.2 8 5.7 Litters affected (#) 4 5 6 2 Total (#) 4 9 13 5 Mean* 0.2 0.4 0.6 0.3 Living pups day 21 p. P. % of males / females (#) 51 / 49 51 / 49 54 / 46 49 / 51 Total 180 164 149 83 Mean* 7.8 7.5 6.8 4.9 Viability index (#) 96 96.9 92.5 84.3 Weaning index (# ) 97.8 94.8 92 94.3

*All mean values were determined in terms of total litter in the respective groups

Applicant's summary and conclusion

Conclusions:

Gavage treatment of male and female Wistar rats with 4-amino-2-hydroxytoluene at dose levels of 40, 200 or 1000 mg/kg bw/day during one generation, revealed parental, reproductive, breeding, and development toxicity at 1000 mg/kg bw/day.

Hence, the definitive parental, reproductive, breeding and developmental No Observed Adverse Effect Level (NOAEL) was established as being 200 mg/kg body weight/day.

Executive summary:

The reproductive toxicity of 4-amino-2-hydroxytoluene was determined following OECD Guideline 415 (One-Generation Reproduction Toxicity Study).

 

The purpose of this study was to assess the effect of 4-amino-2-hydroxytoluene on male and female reproductive performance (such as mating behaviour, conception, parturition, lactation and weaning) when administered by gavage at dose levels of0 (vehicle control), 40, 200 and 1000 mg/kg bw/dayduring one complete reproductive cycle. The study also provided information about the test substance with respect to developmental toxic effects, such as neonatal morbidity, mortality, behaviour and teratogenesis.

 

A total of 192 (96/sex) Wistar rats (source: Charles River Deutschland (Sulzfeld, Germany)), age 5-6 weeks (F0 males); 13-14 weeks (F0 females); weighing173-210 g (F0 males); 215-268 g (F0 females)were housedin groups of 4 animals/sex/cage in suspended stainless steel cages. Animals weremaintained under standard laboratory conditions (temperature:18-24 °C, humidity:30-70 %; 12 h light/12 h dark cycle per d). The animals were acclimated for 9 days and fed onGround Purina Laboratory Chow (ad libitum).

Animals were randomized to the following experimental groups (each consisting of 24/sex) at least 5 days before study start.

- Group 1: 0 mg/kg b.w./day (vehicle control)

- Group 2: 40 mg/kg b.w./day (low dose group)

- Group 3: 200 mg/kg b.w./day (mid dose group)

- Group 4: 1000 mg/kg b.w./day (high dose group)

 

Test substance solutions in vehicle (1% aqueous carboxymethyl cellulose (CMC)) were prepared daily within 4 hours prior to dosing and were homogenized to a visually acceptable level. Experimental animals (males/females of parental generation) were treated according to the following schedule.

 

- Males: 10 weeks prior to mating up to sacrifice.

- Females: 2 weeks prior to mating up to sacrifice.

 

Females were paired on a one-to-one-basis with males from the same treatment group.

 

The day on which mating was designated Day 0 of gestation (= day 0 post-coitum).

 

Each morning following pairing, the trays under the cages were checked for ejected copulation plugs. The day on which a copulation plug was found was designated Day 0 of gestation (= day 0 post-coitum).After 10 days of unsuccessful pairing, male partner was replaced by another proven male. Mated females and males were individually housed in labeled polycarbonate cages containing sawdust as bedding material.

 

Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded. Mating date, identity of male partner, confirmation of pregnancy, and delivery day was recorded for each female.The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). Females that were littering were left undisturbed.

Animals were observed once daily for clinical signs and twice daily for mortality during the entire treatment period. Body weights were recorded on the first day of exposure and weekly thereafter until necropsy. Food consumption was measured at weekly intervals until delivery (except during mating). During lactation, food consumption was recorded on days 1, 4, 7, 14 and 21 post partum.

 

Females without litters or which lost their litters were killed and necropsied. The sex ratio of pups was recorded at Day 0, 4, and Day 21 of lactation. Pup weights were noted on days 1, 4, 7, 14 and 21 of lactation. Pups were also observed daily for survival as well as for physical and behavioural abnormalities. The number of pups was reduced to 4 males and 4 females per litter on day 4 post partum. The remaining pups, from all litters with more than 4 pups per sex, were examined macroscopically. On Day 21 post partum, all pups were examined internally and externally for abnormalities and, if indicated, skeletal development was examined after staining.

 

At necropsy of the parent animals, a macroscopical examination with special focus on the organs of the reproductive system was performed and several organs were weighed.Kidneys and livers from all animals of all groups were processed and examined by the pathologist. All abnormalities were recorded.

 

No treatment-related findings were noted at 40 mg/kg bw/day. In the 1000 mg/kg bw/day and 200 mg/kg bw/day groups, all males and females showed brown discoloration of the urine during treatment.This discoloration was due to the staining properties of the test substance, and thus not considered toxicologically relevant.

 

At 1000 mg/kg bw/day, an increased incidence in mortality (2/24 males (about 8 %) and 5/24 females (about 21%)) was noted. Treatment-related clinical signs in dosed animals consisted of tonic spasms, lateral recumbency, rales, gasping, emaciation, hypothermia, lethargy, labored respiration, pale appearance, chromodacryorrhoea and ptosis.

 

In males, decreased food consumption during pre-mating and decreased body weights during the complete treatment period were observed. In females, decreased food consumption occurred during the complete treatment period and decreased body weight gain during lactation. In males, increased absolute and relative liver weights as well as increased relative weights of kidneys and testes were noted. During the histopathological examination, minor grades of hepatocellular vacuolation were found in females. Males showed increased incidence and severity (primarily slight or moderate) of midzonal/centrilobular liver hypertrophy and increased severity of renal corticomedullary tubular atrophy and hyaline casts.

 

Reproduction parameters like slightly increased mean duration of gestation, delivery difficulties, and a decreased number of pups at birth were observed. In the high dose group, developmental toxicity consisted of an increased post-natal loss resulting in a decreased viability index, as well as an increased incidence of clinical signs and decreased body weights in the offspring. As effects on reproduction, breeding, and development were observed at the same dose levels as general toxicity, a direct effect of the test substance on reproduction cannot be excluded nor established.

Based on the results in this one-generation study,gavage treatment of male and female Wistar rats with 4-amino-2-hydroxytoluene at dose levels of 40, 200 or 1000 mg/kg bw/day, revealed parental, reproductive, breeding, and development toxicity at 1000 mg/kg bw/day.

Hence, the definitive parental, reproductive, breeding and developmental No Observed Adverse Effect Level (NOAEL) was established as being 200 mg/kg body weight/day.

This reproductive toxicity study was classified as acceptable, and satisfies the guideline requirements of OECD 415 method.