Oligomerisation products of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane with acrylic acid and fatty acids, C18-unsatd., dimers and nonanoic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the available weight of evidence from studies with BADGEDA (representing major components of the test substance) and/or nonanoic acid (a metabolite of test substance), the test substance is not expected to pose reproductive or development concern. Nevertheless, as a conservative approach, a LOAEL of 100 mg/kg bw/day (based on lower mean absolute and relative prostate weights (without associated histopathology), reduced sperm motility) from the 90-day repeated dose toxicity study with BADGEDA, has been taken forward for hazard assessment.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From January 15, 2010 to October 13, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Specific details on test material used for the study:

Batch No.: 56728
Purity: 100%

Species:

rat

Strain:

other: Crl:CD (SD)

Details on species / strain selection:

The rat was chosen as the test species because of its acceptance as a predictor of reproductive change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD (SD) strain was used because of the background data available in this laboratory

Sex:

male/female

Details on test animals or test system and environmental conditions:

Acclimatation: five days before treatment
Age at the start of treatment: 9 to 10 weeks
Bodyweights: in the range of 310 to 366g for males and 207 to 244g for females
The temperature: within the range of 19 to 23°C
Relative humidity: 40 to 70%
Artificial lighting: cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours
Feed and water (free access): standard rodent diet (SDS VRF1 Certified) and potable water

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Animals received the test substance or vehicle control formulations orally at a volume-dose of 5 mL/kg bw, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.

Details on mating procedure:

Following two weeks of treatment, males from the toxicity and reproductive subgroups were paired on a one-to-one basis with reproductive subgroup females from within the same treatment group for a period of up to two weeks. Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa and the stage of the oestrous cycle. The day on which evidence of mating was found was designated Day 0 of gestation. Once mating occurred, the males and females were separated and smearing was discontinued. The pre-coital interval was calculated for each female as the time elapsing between initial pairing and detection of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The samples were analysed in accordance with the validated Huntingdon Life Sciences Analytical Procedure. The analytical method involved dissolution and dilution using acetonitrile followed by reverse phase high performance liquid chromatographic analysis with ultra-violet detection (230 nm). Sample concentrations were determined with reference to an external standard prepared at a concentration of 60 µg/mL.
- Concentration in test formulations. At scheduled periods during treatment (first and last weeks), freshly prepared test formulations were sampled (4 × 1 mL, accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate samples were analysed in accordance with the analytical procedure, and the remaining samples were retained for contingency.
- Individual results were within 2% of the mean value, confirming the precision of analysis. The mean concentrations of the test substance in test formulations analysed for the study were within applied limits +10%/-15% of nominal concentrations, confirming accurate formulation, with the exception of one result, Week 1, Day 1, Group 2, 20 mg/mL which was 20.5% below nominal. Additional samples taken from the reformulated Week 1, Day 5, Group 2 formulation were therefore analysed and the achieved concentration was 1.5% above nominal, confirming the accuracy of formulation.

Duration of treatment / exposure:

5 consecutive weeks (two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 7 of lactation).

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

900 mg/kg bw/day (nominal)

No. of animals per sex per dose:

45 males - 65 females
Three groups each comprising five male and five female rats for the toxicity subgroup and five male and ten female rats for the reproductive subgroup received the test substance at doses of 100, 300 or 900 mg/kg bwday.

Control animals:

yes, concurrent vehicle

Details on study design:

The doses used in this study (i.e. 0, 100, 300 and 900 mg/kg bw/day) were selected in conjunction with the Sponsor based on the results of a preliminary study (Huntingdon Life Sciences Study Number: RAJ0001). In that study, dose levels of 80, 250, 750 and 1000 mg/kg bw/day were employed and there was a suggestion of reduced bodyweight gain among males that received 1000 mg/kg bw/day. There was no evidence of an effect of treatment on clinical condition, food consumption, oestrous cycles or organ weights at any dose level investigated, and no treatment-related macroscopic abnormalities were observed. For this study, treatment would continue for approximately 5 weeks and females would be pregnant, therefore in recognition of the potential for reduced bodyweight gain, the high dose level was set at 900 mg/kg bw/day, the low dose of 100 mg/kg bw/day was chosen as an anticipated NOAEL, and the intermediate dose of 300 mg/kg/day at a logarithmic interval between the low and high doses.

Positive control:

-

Parental animals: Observations and examinations:

Clinical observations - Detailed physical examination and arena observations - Sensory reactivity and grip strength - Motor activity
- Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.
- A detailed weekly physical examination including arena observations was performed weekly to monitor general health. For the rerproductive subgroup females during the post-mating period, these observations were conducted on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation.
- Detailed observations were recorded in relation to dose administration. For the toxicity subgroup animals and for reproductive subgroup males these were recorded daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and on one occasion during Week 5. For reproductive subgroup females these were recorded daily during the first week of treatment, twice weekly during Week 2 of treatment, on Days 0, 4, 7, 11, 14 and 20 after mating and Day 4 of lactation.
- Mortality
- Bodyweight - Food and Water consumption
- Haematology, peripheral blood - Blood chemistry
-

Oestrous cyclicity (parental animals):

For 15 days before pairing (including the day of pairing), daily vaginal smears (dry) were taken from all reproductive subgroup females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.

Sperm parameters (parental animals):

Assessment of the testes: tubular stages of the spermatogenic cycle (such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen). Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter.
- Clinical signs: daily records were maintained for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age. Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 and 7 of age. Bodyweight: Individual offspring bodyweights were recorded on Days 1, 4 and 7 of age.

Postmortem examinations (parental animals):

- Detailed necropsy (all animails): tissues (macro and microscopic examinations), external features and orifices, neck and associated tissues, thoracic, abdominal and pelvic cavities and their viscera, organs weight, appearance, and histology.
- Reproductive subgroup females: the number of uterine implantation sites was recorded (+ premature deaths).
The retained tissues were checked before disposal of the carcass.

Postmortem examinations (offspring):

All offspring dying before Day 7 of age were examined as detailed above. The necropsy also included an assessment for the presence of milk in the stomach, where this was possible. The retained tissues were checked before disposal of the carcass.

Statistics:

All statistical analyses were carried out separately for males and females.
- Appropriate group mean values, each with standard deviation (SD) were calculated from individual data.
- Parametric and non-parametric analysis, Fisher’s Exact tests, William's, Dunnetts, and Shirley's test, analysis of covariance, generalised mixed linear model with binomial errors, Wald chi-square test, two-tailed Linear-by-linear test (depending on the end-point).

Reproductive indices:

Assessment of the testes (see above)
Assessment of the ovaries (qualitative evaluation of one section from each ovary was made)
Assessment of the vagina (stage of vaginal oestrous - vaginal epithelial morphology, appearance of the uterus and endometrial glands).

Offspring viability indices:

Post-implantation survival index, live birth index, viability index, and lactation index (%).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No signs were observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. Signs relating to dose administration were restricted to a dose-related increased incidence of chin rubbing on return to the home cage among males and females at 300 or 900 mg/kg bw/day, with associated salivation or post-salivation staining also recorded for occasional animals. Occasional reproductive subgroup females in the 100 mg/kg bw/day showed these signs during the gestation period. These findings are commonly observed following oral (gavage) administration and are considered to reflect distaste of the test formulation rather than a direct effect of treatment

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One reproductive subgroup male receiving 100 mg/kg bw/day was found dead prior to dose administration on Day 34 of the study. Ante mortem signs for this animal were restricted to noisy respiration (rales) on the previous day and there were no macroscopic abnormalities detected. In the absence of any other mortalities in this or higher dose groups, the demise of this male in the lowest dose group was considered incidental and unrelated to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

It was noted that during the first two weeks of treatment, mean weight gain in all treated groups was lower than control, and between Weeks 2 and 5 the weight gain of toxicity subgroup females was higher than control, with all differences attaining statistical significance. There was, however, no dose response apparent, and therefore these differences were considered to be of no toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Assessment of haematological parameters for toxicity subgroup animals during Week 5 of treatment revealed slightly prolonged prothrombin time among males and females receiving 900 or 300 mg/kg bw/day, with a dose-related trend apparent and statistical significance attained. The time for males receiving 900 mg/kg bw/day was marginally outside the 5-95% confidence limits of general Historical Control Data (HCD) for rats of this age and strain but males in the 300 mg/kg bw/day were within the range, as were the females in the 300 or 900 mg/kg bw/day groups.
- At 900 or 300 mg/kg bw/day, group mean activated partial thromboplastin time was slightly shortened compared with the concurrent controls. A dose dependent trend was apparent, however statistical significance was not attained, and at 900 mg/kg bw/day the group mean time for males was influenced by one atypical animal with a particularly short clotting time.
- Some other minor inter-group differences in haematological parameters attained statistical significance, but no clear evidence of an adverse effect of treatment. Among all groups of treated males, the mean cell haemoglobin concentration was higher than control, with a dose-response apparent. Values were within the 5-95% confidence limits of general HCD in the 100 and 300 mg/kg bw/day groups, and only marginally outside these limits by 0.1g/dL in the 900 mg/kg bw/day group, and in the absence of any other changes in erythrocytic parameters, or a similar effect among females, these differences were considered likely to be fortuitous and unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry:
During Week 5, total bilirubin and total bile acid concentrations were statistically significantly high in males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day. Total bilirubin concentrations for animals in the 900 mg/kg bw/day group were outside the 5-95% confidence limits of general HCD for rats of this age and strain; no HCD are available for total bile acid concentrations. In addition, cholesterol concentrations were statistically significantly high in males and females in all treated groups with a dose related trend apparent; concentrations for males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day were outside the 5-95% confidence limits of general HCD. All other inter-group differences from controls (such as potassium concentrations at 900 or 300 mg/kg bw/day) were either minor, showed no consistency across the sexes or lacked dose-relationship and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

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):

NB. Sensory reactivity observations and grip strength:
Males receiving 900 mg/kg bw/day showed low forelimb grip strength values compared with controls (p<0.05). Two of the control values (1.81 and 1.82 kg) were, however, considerably higher than all other values in the control and treated groups whereas values for the other control males were similar to those of males in all of the treated groups. These differences were therefore attributed to two atypically high control group values rather than treatment. Hindlimb values for males and forelimb and hindlimb values for females were similar in all groups.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

During initial microscopical examination, lungs from 3 toxicity subgroup males receiving 900 mg/kg bw/day had minimal aggregations of alveolar macrophages. In addition, the kidney from one male in this group had minimal multiple cortical scarring, minimal focal cortical tubular basophilia and cortical tubular dilatation. To assess the toxicological significance of this finding, a trackdown was carried out on lungs and kidney from toxicity and reproductive subgroup males given 100 or 300 mg/kg bw/day test substance. Subsequent microscopical examination showed no dose dependent trend in severity or incidence level for these findings and hence they were considered as incidental. The incidence and distribution of all the other findings were consistent with the common background for animals of this age at these laboratories.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity scores for toxicity subgroup males and females showed some inter-group variation however no dose response was apparent and no association with treatment was inferred.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycle length and pre-coital interval of the reproductive subgroup females was unaffected by treatment at all dose levels investigated. All animals mated at the first oestrus opportunity after cohabitation.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Seminiferous tubules of all males of both the toxicity and reproductive subgroups were evaluated with respect to their stage in the spermatogenic cycle and integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance and fertility of the reproductive subgroup females was unaffected by treatment at all dose levels investigated.

Additional data:
There was no effect of treatment at any dose level investigated on the gestation length of reproductive subgroup females. There were no instances of dystocia and the gestation index was 100% in all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: the test substance was well tolerated

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no effect of parental treatment at any dose level investigated on the mean number of implantation sites or litter size. Post-natal survival was unaffected by treatment, and the percentage of males in the litters in all groups was consistent between Days 1 and 7 of age, indicating that there was no preferential mortality of either sex.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

There were no macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment with the test substance.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: The test substance was well tolerated

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the study conditions, the NOAEL for systemic toxicity in rats following 5 weeks of treatment was 900 mg/kg bw/day of the test substance. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be 900 mg/kg bw/day.

Executive summary:

A study was conducted to determined the toxicity to reproduction of the test substance according to OECD Guideline 422. The general systemic toxic potential of the substance to Crl:CD (SD) rats was assessed by oral administration (gavage) over a period of at least five weeks. Three groups each comprising five male and five female rats for the toxicity subgroup and five male and ten female rats for the reproductive subgroup received the test substance at doses of 0, 100, 300 or 900 mg/kg bw/day. Toxicity subgroup males and females and reproductive subgroup males were treated daily for five consecutive weeks. Reproductive subgroup females were treated daily for two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 7 of lactation. A similarly constituted control group received the vehicle, propylene glycol, at the same volume-dose. During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology, blood chemistry, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

There was one mortality during the course of the study. One reproductive subgroup male receiving 100 mg/kg bw/day was found dead prior to dose administration on Day 34 of the study. Ante mortem signs were restricted to noisy respiration (rales) on the previous day, and there were no macroscopic abnormalities detected. In the absence of any other mortalities, the demise of this animal was considered incidental and unrelated to treatment. There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. At 900 mg/kg bw/day, mean bodyweight gain of males was slightly lower than control during the first week of treatment. During the second week, weight gain for these males remained slightly (but not significantly) lower than control, but for the first two weeks overall the difference remained significant. Thereafter, mean weight gain was essentially similar to control in all treated groups, and the bodyweight gain of females during gestation and lactation was unaffected by treatment at all dose levels investigated. There was no effect of treatment on the food consumption of males and females throughout the study. Sensory reactivity findings, grip strength values and motor activity for toxicity subgroup males and females during Week 5 of treatment were considered to be unaffected by treatment. The haematological and biochemical investigation for toxicity subgroup animals after five weeks of treatment did not reveal any changes that were clearly attributable to treatment. Haematological assessment revealed slightly prolonged prothrombin time among males and females receiving 900 or 300 mg/kg bw/day. Biochemical changes in the blood plasma comprised high total bilirubin and total bile acid concentrations in males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day, and high cholesterol concentrations in males and females in all treated groups. A dose-related trend was evident for all of these haematological and biochemical parameters. Despite these differences, suggestive of changes in liver function, there was no evidence of changes in clinical condition of the animals or of macroscopic or microscopic hepatotoxicity, therefore the differences in prothrombin time, cholesterol and biliary parameters were considered unlikely to be of toxicological significance. There was no clear evidence of an adverse effect of treatment on mean unadjusted or adjusted organ weights among toxicity or reproductive subgroup animals at scheduled termination. Among the Toxicity subgroup females, adrenal weights were slightly low in all treated groups, however there were no associated macroscopic/microscopic abnormalities in this organ to explain the differences in weight. There were no macroscopic abnormalities detected at scheduled termination of the toxicity or reproductive subgroup animals that were considered to represent an adverse effect of treatment. The microscopic examination performed after five weeks of treatment revealed no test substance-related lesions. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted. Oestrous cycle length, pre-coital interval, mating performance and fertility and gestation length of the reproductive subgroup females was unaffected by treatment at all dose levels investigated. There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment, and offspring survival and growth from birth to Day 7 of age was unaffected by treatment. There were no macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment with the test substance. Treatment with the test substance at doses up to and including 900 mg/kg bw/day was well tolerated. Under the study conditions, the NOAEL for systemic toxicity in rats following 5 weeks of treatment was 900 mg/kg bw/day of the test substance. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be 900 mg/kg bw/day (Stannard, 2010).

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

This endpoint is read across from 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid; Refer to read-across justification appended in Section 13. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

No. of animals per sex per dose:

10 animals/group

Reproductive performance:

no effects observed

No effects were noted on implantations, length of gestation, proportion of dams delivering at least one live pup or pup viability. Pup weights were reduced on day 4 post parturition in the high-dose group. No malformations or gross lesions were seen in pups at any dose level.

Key result

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Due to presence of increased incidence of respiratory rales even at the lowest dose

Remarks on result:

other: Systemic effects

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects were noted on implantations, length of gestation, proportion of dams delivering at least one live pup or pup viability.

Remarks on result:

other: Reproductive and developmental effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Mortality occurred in the highdose group (3 of 10 rats) and mid-dose group (1 of 10 rats). In the 1000 and 2000 mg/kg-bw/day dose groups, significant increases in the incidence of rales (p <Mortality occurred in the highdose group (3 of 10 rats) and mid-dose group (1 of 10 rats). In the 1000 and 2000 mg/kg-bw/day dose groups, significant increases in the incidence of rales (p < 0.01) and excess salivation (p < 0.01) were reported during premating and gestation. High-dose females also exhibited lethargy, unkempt coats and labored breathing. In the highdose group, food consumption was reduced throughout the study, average maternal body weights were decreased on days 10 – 16 of gestation and body weight gains were significantly (p < 0.05) reduced. The high-dose group showed reduced mating and fertility that were related to mortality. However, the duration of cohabitation and fertility and gestation indices were comparable to controls. Rats in the 200 mg/kg-bw/day dose group also showed clinical signs, including a significant increase (p < 0.01) in the incidence of respiratory rales during pre-mating and gestation but not during lactation. No effects were noted on implantations, length of gestation, proportion of dams delivering at least one live pup or pup viability. Pup weights were reduced on day 4 post parturition in the high-dose group. No malformations or gross lesions were seen in pups at any dose level0.01) and excess salivation (p < 0.01) were reported during premating and gestation. High-dose females also exhibited lethargy, unkempt coats and labored breathing. In the highdose group, food consumption was reduced throughout the study, average maternal body weights were decreased on days 10 – 16 of gestation and body weight gains were significantly (p < 0.05) reduced. The high-dose group showed reduced mating and fertility that were related to mortality. However, the duration of cohabitation and fertility and gestation indices were comparable to controls. Rats in the 200 mg/kg-bw/day dose group also showed clinical signs, including a significant increase (p < 0.01) in the incidence of respiratory rales during pre-mating and gestation but not during lactation. No effects were noted on implantations, length of gestation, proportion of dams delivering at least one live pup or pup viability. Pup weights were reduced on day 4 post parturition in the high-dose group. No malformations or gross lesions were seen in pups at any dose level

Conclusions:

The test substance did not have any adverse effects on reproductive or development parameters. The NOAEL was established at the highest tested dose of 2,000 mg/kg bw/day.

Executive summary:

In a reproductive/developmental toxicity study, female Sprague-Dawley rats (10/group) were administered read across substance heptanoic acid via gavage (in corn oil) at doses of 0, 200, 1,000 or 2,000 mg/kg bw/day for one week prior to a 7-day cohabitation period through gestation, parturition and a 4-day postpartum period. Under the conditions of the study, the LOAEL for systemic toxicty was established at 200 mg/kg bw/day based on increased incidence of respiratory rales; and the NOAEL for reproductive and development toxicity was established at 2,000 mg/kg bw/day. The same NOAEL and LOAELs can be extrapolated for the SoI. As explained in the read across justification, the differences in the structure of the components between the SoI and the read across substance are unlikely to lead to differences in toxicities in the study that are higher than typical experimental error of the test method.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

This endpoint is read across from 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid; Refer to read-across justification appended in Section 13. The screening study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No signs were observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. Signs relating to dose administration were restricted to a dose-related increased incidence of chin rubbing on return to the home cage among males and females at 300 or 900 mg/kg bw/day, with associated salivation or post-salivation staining also recorded for occasional animals. Occasional reproductive subgroup females in the 100 mg/kg bw/day showed these signs during the gestation period. These findings are commonly observed following oral (gavage) administration and are considered to reflect distaste of the test formulation rather than a direct effect of treatment

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One reproductive subgroup male receiving 100 mg/kg bw/day was found dead prior to dose administration on Day 34 of the study. Ante mortem signs for this animal were restricted to noisy respiration (rales) on the previous day and there were no macroscopic abnormalities detected. In the absence of any other mortalities in this or higher dose groups, the demise of this male in the lowest dose group was considered incidental and unrelated to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

It was noted that during the first two weeks of treatment, mean weight gain in all treated groups was lower than control, and between Weeks 2 and 5 the weight gain of toxicity subgroup females was higher than control, with all differences attaining statistical significance. There was, however, no dose response apparent, and therefore these differences were considered to be of no toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Assessment of haematological parameters for toxicity subgroup animals during Week 5 of treatment revealed slightly prolonged prothrombin time among males and females receiving 900 or 300 mg/kg bw/day, with a dose-related trend apparent and statistical significance attained. The time for males receiving 900 mg/kg bw/day was marginally outside the 5-95% confidence limits of general Historical Control Data (HCD) for rats of this age and strain but males in the 300 mg/kg bw/day were within the range, as were the females in the 300 or 900 mg/kg bw/day groups.
- At 900 or 300 mg/kg bw/day, group mean activated partial thromboplastin time was slightly shortened compared with the concurrent controls. A dose dependent trend was apparent, however statistical significance was not attained, and at 900 mg/kg bw/day the group mean time for males was influenced by one atypical animal with a particularly short clotting time.
- Some other minor inter-group differences in haematological parameters attained statistical significance, but no clear evidence of an adverse effect of treatment. Among all groups of treated males, the mean cell haemoglobin concentration was higher than control, with a dose-response apparent. Values were within the 5-95% confidence limits of general HCD in the 100 and 300 mg/kg bw/day groups, and only marginally outside these limits by 0.1g/dL in the 900 mg/kg bw/day group, and in the absence of any other changes in erythrocytic parameters, or a similar effect among females, these differences were considered likely to be fortuitous and unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry:
During Week 5, total bilirubin and total bile acid concentrations were statistically significantly high in males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day. Total bilirubin concentrations for animals in the 900 mg/kg bw/day group were outside the 5-95% confidence limits of general HCD for rats of this age and strain; no HCD are available for total bile acid concentrations. In addition, cholesterol concentrations were statistically significantly high in males and females in all treated groups with a dose related trend apparent; concentrations for males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day were outside the 5-95% confidence limits of general HCD. All other inter-group differences from controls (such as potassium concentrations at 900 or 300 mg/kg bw/day) were either minor, showed no consistency across the sexes or lacked dose-relationship and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

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):

NB. Sensory reactivity observations and grip strength:
Males receiving 900 mg/kg bw/day showed low forelimb grip strength values compared with controls (p<0.05). Two of the control values (1.81 and 1.82 kg) were, however, considerably higher than all other values in the control and treated groups whereas values for the other control males were similar to those of males in all of the treated groups. These differences were therefore attributed to two atypically high control group values rather than treatment. Hindlimb values for males and forelimb and hindlimb values for females were similar in all groups.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

During initial microscopical examination, lungs from 3 toxicity subgroup males receiving 900 mg/kg bw/day had minimal aggregations of alveolar macrophages. In addition, the kidney from one male in this group had minimal multiple cortical scarring, minimal focal cortical tubular basophilia and cortical tubular dilatation. To assess the toxicological significance of this finding, a trackdown was carried out on lungs and kidney from toxicity and reproductive subgroup males given 100 or 300 mg/kg bw/day test substance. Subsequent microscopical examination showed no dose dependent trend in severity or incidence level for these findings and hence they were considered as incidental. The incidence and distribution of all the other findings were consistent with the common background for animals of this age at these laboratories.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity scores for toxicity subgroup males and females showed some inter-group variation however no dose response was apparent and no association with treatment was inferred.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycle length and pre-coital interval of the reproductive subgroup females was unaffected by treatment at all dose levels investigated. All animals mated at the first oestrus opportunity after cohabitation.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Seminiferous tubules of all males of both the toxicity and reproductive subgroups were evaluated with respect to their stage in the spermatogenic cycle and integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance and fertility of the reproductive subgroup females was unaffected by treatment at all dose levels investigated.

Additional data:
There was no effect of treatment at any dose level investigated on the gestation length of reproductive subgroup females. There were no instances of dystocia and the gestation index was 100% in all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: the test substance was well tolerated

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no effect of parental treatment at any dose level investigated on the mean number of implantation sites or litter size. Post-natal survival was unaffected by treatment, and the percentage of males in the litters in all groups was consistent between Days 1 and 7 of age, indicating that there was no preferential mortality of either sex.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

There were no macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment with the test substance.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: The test substance was well tolerated

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the study conditions, the NOAEL for systemic toxicity and for the reproductive/developmental toxicity were both concluded to be 900 mg/kg bw/day.

Executive summary:

A study was conducted to determined the toxicity to reproduction of the read across substance (4,4'-Isopropylidenediphenol oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid; i.e., BADGEDA) according to OECD Guideline 422. Treatment with the test substance at doses up to and including 900 mg/kg bw/day was well tolerated (Stannard, 2010). Therefore, based on the study results of the read across substance, the NOAEL for systemic toxicity and for the reproductive/developmental toxicity of the SoI can be concluded to be at 900 mg/kg bw/day. As explained in the read across justification, the differences in the structure of the components between the SoI and the read across substance are unlikely to lead to differences in toxicities in an combined reproductive and development toxicity screening assay that are higher than typical experimental error of the test method.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Well-conducted guideline compliant study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In absence of a study with the test substance, its toxicity potential has been assessed on the basis of available reproductive and development studies on the major components and metabolites.

 

A study was conducted to determine the toxicity to reproduction of the read-across substance, 4,4’-isopropylidenediphenol oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid or BADGEDA. The general systemic toxic potential of the test substance to Crl:CD (SD) rats was assessed by oral administration (gavage) over a period of at least five weeks. Three groups of 5 male and 5 female rats for the toxicity subgroup and 5 male and 10 female rats for the reproductive subgroup received the test substance at doses of 0, 100, 300 or 900 mg/kg bw/day. Toxicity subgroup males and females and reproductive subgroup males were treated daily for five consecutive weeks. Reproductive subgroup females were treated daily for two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 7 of lactation. A similarly constituted control group received the vehicle, propylene glycol, at the same volume-dose. During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology, blood chemistry, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken. There was one mortality during the course of the study. One reproductive subgroup male receiving 100 mg/kg bw/day was found dead prior to dose administration on Day 34 of the study. Ante mortem signs were restricted to noisy respiration (rales) on the previous day, and there were no macroscopic abnormalities detected. In the absence of any other mortality, the demise of this animal was considered incidental and unrelated to treatment. There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. At 900 mg/kg bw/day, mean bodyweight gain of males was slightly lower than control during the first week of treatment. During the second week, weight gain for these males remained slightly (but not significantly) lower than control, but for the first two weeks overall the difference remained significant. Thereafter, mean weight gain was essentially similar to control in all treated groups, and the bodyweight gain of females during gestation and lactation was unaffected by treatment at all dose levels investigated. There was no effect of treatment on the food consumption of males and females throughout the study. Sensory reactivity findings, grip strength values and motor activity for toxicity subgroup males and females during Week 5 of treatment were considered to be unaffected by treatment. The haematological and biochemical investigation for toxicity subgroup animals after five weeks of treatment did not reveal any changes that were clearly attributable to treatment. Haematological assessment revealed slightly prolonged prothrombin time among males and females receiving 900 or 300 mg/kg bw/day. Biochemical changes in the blood plasma comprised high total bilirubin and total bile acid concentrations in males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day, and high cholesterol concentrations in males and females in all treated groups. A dose-related trend was evident for all of these haematological and biochemical parameters. Despite these differences, suggestive of changes in liver function, there was no evidence of changes in clinical condition of the animals or of macroscopic or microscopic hepatotoxicity, therefore the differences in prothrombin time, cholesterol and biliary parameters were considered unlikely to be of toxicological significance. There was no clear evidence of an adverse effect of treatment on mean unadjusted or adjusted organ weights among toxicity or reproductive subgroup animals at scheduled termination. Among the Toxicity subgroup females, adrenal weights were slightly low in all treated groups, however there were no associated macroscopic/microscopic abnormalities in this organ to explain the differences in weight. There were no macroscopic abnormalities detected at scheduled termination of the toxicity or reproductive subgroup animals that were considered to represent an adverse effect of treatment. The microscopic examination performed after five weeks of treatment revealed no test substance-related lesions. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted. Oestrous cycle length, pre-coital interval, mating performance and fertility and gestation length of the reproductive subgroup females was unaffected by treatment at all dose levels investigated. There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment, and offspring survival and growth from birth to Day 7 of age was unaffected by treatment. There were no macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment with the test substance. Treatment with the test substance at doses up to and including 900 mg/kg bw/day was well tolerated. Under the study conditions, the NOAEL for systemic toxicity in rats following 5 weeks of treatment was 900 mg/kg bw/day of the test substance. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be 900 mg/kg bw/day (Stannard, 2010).

 

Further, a 90-day repeated dose toxicity study conducted with the read across substance, BADGEDA, has also been evaluated to assess the reproductive toxicity potential of the test substance.

 

The 90-day repeated dose toxicity study of the read across substance to Wistar rats was investigated in a GLP-compliant dose-effect study according to the OECD TG 408 (1998) and EU B.29 (1996) protocols. The test substance was administered daily by oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day (named groups 1 to 4, respectively) using a volume of 5 mL/kg body weight. The control group was treated with the vehicle, PEG 300, only. The groups comprised 10 animals per sex which were sacrificed after 92/93 days of treatment. Apart from the standard repeated dose parameters, the study included evaluation of organ weights, histological examinations of organs inclusive of gonads, analysis of sperm sample to assess count, motility and morphology and stages of oestrous and spermatogenesis and histopathology of the interstitial cell structure.

The administration of the test substance to the test animals resulted in no test substance-related deaths, no differences in mean food consumption, weekly observations (weeks 1 - 13) or functional observational battery (week 13), no differences of toxicological relevance in the fore- and hind limb grip strength values, no test substance-related differences in the ophthalmoscopy and no test substance-related effects on urine parameters.

Test substance-related clinical signs included salivation in both sexes treated with 300 mg/kg bw/day and 1000 mg/kg bw/day. This finding was first noted in week 5 and was frequently seen throughout the rest of the study. This finding is generally associated with a bitter taste of the dosing solutions and is a secondary, non-adverse effect. Clearly lower mean body weight development was noted from Day 15 of treatment in males treated with 1000 mg/kg bw/day.

Reduced locomotor activity was noted in males at all dose levels and in females treated at 1000 mg/kg bw/day.

In males treated with 1000 mg/kg bw/day, higher relative neutrophil and monocyte counts, lower mean relative lymphocyte count and lower platelet count were noted when compared with the controls. Females treated with 1000 mg/kg bw/day had elevated mean relative monocyte counts, reduced platelets and an increased mean absolute neutrophil count. Males and females treated with 300 mg/kg bw/day had only lower mean platelet counts when compared with the controls. Males and females treated with 100 mg/kg bw/day were unaffected.

Test substance-related differences in clinical biochemistry parameters were noted in males at all dose levels. Increased mean cholesterol and mean phospholipid levels were noted in males at all dose levels, and is often associated with changes in lipid metabolism. Males also showed increased enzyme activation of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase. Similar findings were seen in females treated with 1000 mg/kg bw/day: increased activities of the aspartate and alanine aminotransferases. These differences, while test substance related, are generally considered to be metabolic adaptation and of no toxicological relevance. However, parameters that were reduced in males at 1000 mg/kg bw/day included glucose levels, potassium levels, protein and globulin levels. Although these values remained within the limits of the historical control values, they were considered to be related to the treatment with the test substance. In females, significantly higher cholesterol and phospholipid levels (both p<0.01) were seen at 1000 mg/kg/day and 300 mg/kg/day, exceeded the upper limits of the historical control data and were considered to be test substance-related.

The reduction of sperm motility noted at all dose levels was considered to be related to the test substance. There were no test substance-related effects in the testis, the integrity of the various cell types present within the different stages of the sperm cycle (spermatogenesis), the quantification of the different stages (quantitative staging) and the integrity of interstitial cells being unaffected. 

Lower mean absolute and relative prostate weights noted at 100, 300 and 1000 mg/kg/day, and higher mean relative kidney weights in males at 300 mg/kg/day and 1000 mg/kg/day were considered to be test substance-related effects. Lower mean absolute thymus weight, mean thymus-to body weight ratio and mean thymus-to-brain weight ratio in females at 1000 mg/kg bw/day were considered to be a stress reaction.

The test-substance induced microscopic findings in the liver of a few rats at 1000 mg/kg bw/day (minimal hepatocellular necrosis, moderate hepatocellular hypertrophy/vacuolation); they were considered to be the histological correlates of the increased liver enzymes recorded in clinical biochemistry. The most prominent liver microscopic findings were observed in the female rat no. 72 (1000 mg/kg bw/day) and were associated with changes in kidneys, lymphoid organs and ovaries consistent with stress/altered metabolic status.

The pathogenesis of the multifocal cell hypertrophy observed in the pars distalis of the pituitary gland of males cannot be ascertained. However, the pituitary findings and the decreased prostate/seminal vesicles weights may represent an indicator of mild disruption of testosterone production/levels.

The pathogenesis underlying the decreased lymphocytes in the mesenteric lymph nodes is unknown in the absence of changes in the other lymphoid organs.

In conclusion,based on lower mean absolute and relative prostate weights (without associated histopathology), reduced sperm motility, increased cholesterol and phospholipid levels, locomotor activity reductions, no-observed adverse effect levels (NOAEL) for systemic effects were below the lowest dose level of 100 mg/kg bwday (i.e., only a LOAEL was derived).

 

Further, the toxicity potential of the hydrolytic product/metabolite, nonanoic acid has been evaluated on the basis of an available study with a worst case shorter chain fatty acid i.e., heptanoic acid, published in the US EPA screening level hazard characterisation document on C7 -C9 aliphatic aldehydes and carboxylic acids in 2007.

In a reproductive/developmental toxicity study, female Sprague-Dawley rats (10/group) were administered heptanoic acid via gavage (in corn oil) at doses of 0, 200, 1000 or 2000 mg/kg-bw/day for one week prior to a 7-day cohabitation period through gestation, parturition and a 4-day postpartum period. Mortality occurred in the high-dose group (3 of 10 rats) and mid-dose group (1 of 10 rats). In the 1,000 and 2,000 mg/kg bw/day dose groups, significant increases in the incidence of rales (p < 0.01) and excess salivation (p < 0.01) were reported during premating and gestation. High-dose females also exhibited lethargy, unkempt coats and labored breathing. In the high dose group, food consumption was reduced throughout the study, average maternal body weights were decreased on days 10 – 16 of gestation and body weight gains were significantly (p < 0.05) reduced. The high-dose group showed reduced mating and fertility that were related to mortality. However, the duration of cohabitation and fertility and gestation indices were comparable to controls. Rats in the 200 mg/kg bw/day dose group also showed clinical signs, including a significant increase (p < 0.01) in the incidence of respiratory rales during pre-mating and gestation but not during lactation. No effects were noted on implantations, length of gestation, proportion of dams delivering at least one live pup or pup viability. Pup weights were reduced on day 4 post parturition in the high-dose group. No malformations or gross lesions were seen in pups at any dose level. Therefore, no NOAEL for systemic toxicity could be established due to presence of increased incidence of respiratory rales even at the lowest dose. Nevertheless, the NOAEL for reproductive and development toxicity was established at 2,000 mg/kg bw/day.

 

Overall, based on the available weight of evidence from studies with BADGEDA (representing major components of the test substance) and/or nonanoic acid (a metabolite of test substance), the test substance is not expected to pose reproductive or development concern. Nevertheless, as a conservative approach, a LOAEL of 100 mg/kg bw/day (based on lower mean absolute and relative prostate weights (without associated histopathology), reduced sperm motility) from the 90-day repeated dose toxicity study with BADGEDA, has been taken forward for hazard assessment.

 

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the occurrence of reduced sperm motility in the absence of correlating or any other adverse effects on other sperm parameters or the spermatogenesis stages in the 90-day repeated dose study with read across substance, BADGEDA, no classification is required for this endpoint according to CLP (1272/2008) criteria. This is further supported by the absence of any adverse effects in the combined repeated dose toxicity and reproductive/developmental screening study (OECD 422) conducted with BADGEDA as well as reproductive/developmental toxicity studies with heptanoic acid (which is structurally similar to the non-overlapping metabolite, nonanoic acid).