Benzenamine, N-phenyl-, styrenated

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction: Screening study, oral: gavage, Sprague-Dawley rat m/f, 10/sex/dose, 0, 50, 250, 600 mg/kg bw/d (OECD 422, GLP):

NOAEL = 600 mg/kg bw/d (systemic toxicity, P generation, only adaptive, non-adverse responses)

NOAEL = 250 mg/kg bw/d (effects on fertility, based on pre-implantation losses, possibly secondary effects)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-02-21 - 2005-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for Testing of Chemicals, No. 422: “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

n/a

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Sprague-Dawley Crl:CD (SD) IGS BR strain rats

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately six weeks (P generation)
- Weight at study initiation: At the start of treatment, the males weighed 184 to 239g, the females weighed 145 to 188g. (P generation)
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase, animals were transferred to similar cages on an one male : one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd. Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd, Hull, UK) and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet (e.g. ad libitum): A pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK) was used throughout the treatment period ad libitum.
- Water (e.g. ad libitum): Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage.
- Acclimation period: On receipt, the animals were examined for signs of ill-health or injury. The animals were acclimatised for up to ten days, during which time their health status was assessed.

DETAILS OF FOOD AND WATER QUALITY:
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
The animals were housed in air-conditioned rooms within the Laboratories Limited Barrier Maintained Rodent Facility
- Temperature (°C) / Humidity (%): Environmental conditions were continuously monitored by a computerised system and print-outs of hourly mean temperatures and humidities are included in the study records. The temperature and relative humidity controls were set to achieve target values of 21±2°C and 55 ±15% respectively. Occasional deviations from these targets were considered not to affect the purpose or integrity of the study.
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of the study, the test material was prepared at the appropriate concentrations as a solution in Corn Oil. The stability and homogeneity of the test material formulations were
determined. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4ºC in the dark.

VEHICLE
- Concentration in vehicle: 0, 12.5, 62.5, or 150 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating.
- After successful mating each pregnant female was caged (how): Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material formulations were determined by the Analytical Laboratory. Results are given in in the attachment and show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4°C in the dark.
Samples were taken of each test material formulation and were analysed for concentration of CAS No 68442-68-2 at the Analytical Laboratory. The method used for analysis of formulations and the results obtained are given in the attachment. The results indicate that the prepared formulations were within acceptable limits for the purpose of this study.

Duration of treatment / exposure:

Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.

Frequency of treatment:

daily

Details on study schedule:

Chronological Sequence of Study
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).
ii) Prior to the start of treatment and once weekly, all animals were observed for signs of functional/behavioural toxicity.
iii) One day prior to pairing (Day 14), blood samples were taken from five males and five females, randomly selected from each dose group and analysed for haematological and blood chemical assessment.
iv) On Day 15, all animals were paired on a 1 male : 1 female basis within each dose group for a maximum of fourteen days.
v) Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
vi) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vii) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean pup weight, clinical observations and landmark developmental signs were also performed during this period.
viii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
ix) Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically on Day 43.
x) At Day 5 post partum, all surviving females and offspring were killed and examined macroscopically.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 / sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of a preliminary range-finder.
- Rationale for animal assignment (if not random): The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories.

Positive control:

not required

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity (except for females which did not achieve pregnancy). Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed: Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behaviour, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation,

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to the start of treatment) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt change.

OTHER: Yes, see chapter "repeated dose toxicity" for detailed examinations with regard to overall systemic toxicity

Oestrous cyclicity (parental animals):

During mating, a vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded.

Sperm parameters (parental animals):

Parameters examined in the male parental generation: testis weight, epididymis weight, sperm count in testes, sperm count in epididymides

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
For each litter the following was recorded:
i) Number of offspring born
ii) Number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum
iii) Clinical condition of offspring from birth to Day 4 post partum
iv) Individual offspring and litter weights on Day 1 and 4 post partum
All live offspring were observed for the detachment of pinna and assessed for reflexological response to stimuli by assessing surface righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
Yes. All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43.
- Maternal animals: Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.

GROSS PATHOLOGY: Yes
The corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. In the case of non-pregnant females, the procedure was enhanced by staining the uteri with a 1% ammonium polysulphide solution.
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
The following organs, removed from adult animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus

HISTOPATHOLOGY
Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin. The tissues marked with x were also removed from the remaining animals:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulating gland (x)
Colon
Duodenum
Epididymides * (x)
Eyes
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi) #
Lymph nodes (cervical and mesenteric)
Mammary gland
Muscle (skeletal)
Ovaries (x)
Pancreas
Pituitary (x)
Prostate (x)
Oesophagus
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles (x)
Skin (hind limb)
Spinal cord (cervical)
Spleen
Stomach
Thyroid/parathyroid
Trachea
Testes * (x)
Thymus
Urinary bladder
Uterus/Cervix (x)
Vagina

* = preserved in Bouin’s fluid and then in 70% IMS after forty-eight hours
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

The tissues from five selected control and 600 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues marked with x from the remaining control and 600 mg/kg/day were also processed.
Since there were indications of treatment-related changes in the liver and thyroid glands examination was subsequently extended to include similarly prepared sections of liver and thyroid gland tissues from five selected males and females from the 250 and 50 mg/kg/day dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Postmortem examinations (offspring):

SACRIFICE
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Treatment of Data
Data were processed to give group mean values and standard deviations where appropriate.
For bodyweights and food consumptions during gestation, group mean values were calculated using data from females which produced live young.
For bodyweights and food consumptions during lactation, group mean values were calculated using data from females with live young at Day 5 of lactation.

Statistical Analysis
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.
Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p >0.05 (not significant)

Reproductive indices:

Reproductive Indices
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Number of animals paired) * 100
Pregnancy Index (%) = (Number of pregnant females / number of animals mated) * 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of animals delivering live offspring / Number of pregnant females) * 100

Offspring viability indices:

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i. Implantation Losses (%)
Group mean percentile pre-implantation and post implantation loss were calculated for each female/litter as follows:
% pre - implantation loss = ((Number of Corpora Lutea - Number of implantation sites) / Number of corpora lutea) * 100
% Post-implantation loss = ((Number of implantation sites – Total number of offspring born) / Number of implantation sites) * 100

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) * 100
Viability Index (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) * 100

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4 and 13 post partum, using the following formula: (Number of male offspring / Total number of offspring) * 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinically observable signs of toxicity were detected.
Transient episodes of increased salivation were detected soon after dosing for animals of either sex treated with 600 mg/kg/day from Day 5, with the observation also evident up to one hour after dosing for males. Isolated incidents of increased salivation were also apparent at 250 mg/kg/day, during the later stages of the study. This observation is often reported following the oral administration of a slightly unpalatable or irritant test material formulation, and in isolation, is considered to be of no toxicological importance.
Incidents of fur loss, scab formation and staining of the external body surface, detected throughout the treatment groups, are common low incidence findings in laboratory maintained animals and are considered unrelated to treatment.
One female treated with 50 mg/kg/day displayed damage to the tail tip and one control male displayed an injury to the eye. These were physical injuries and unrelated to treatment. Additionally, one control female displayed an incident of diuresis.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no deaths related to treatment.
One female treated with 250 mg/kg/day was killed in extremis on gestation Day 25, following difficulties encountered during parturition. In isolation and in the absence of any histopathological correlates, this death was considered unrelated to treatment.
There were no further deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effect on bodyweight change was detected for treated males in comparison to controls throughout the treatment period, or for females throughout the maturation, gestation or lactation phases of the study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effect on dietary intake was detected for males throughout the treatment period, or for females during maturation and gestation. Food efficiencies were unaffected.
A slight reduction in dietary intake was evident for females treated with 600 mg/kg/day during lactation, compared with controls, however differences failed to attain statistical significance. Lower litter size was apparent at this dose level and it is considered that the observed differences in food intake probably reflected the lower litter demand on the parental females rather than any adverse effect of treatment.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles revealed no intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related haematological changes were detected prior to mating.
Females treated with 600 mg/kg/day showed a statistically significant reduction in mean cell volume (MCV) when compared with control females. The significance was minimal (p<0.05) and in the absence of supporting data to suggest this was related to treatment, this finding was considered to have arisen incidentally.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 600 mg/kg/day showed slightly higher alkaline phospatase levels than control males (p<0.05). Additionally, reduced cholesterol levels were detected for males treated with 600 and 250 mg/kg/day in comparison to controls (p<0.05 and p<0.01, respectively).
The statistically significant reductions in urea levels detected for males treated with 600 mg/kg/day were of minimal significance (p<0.05) and in the absence of any histopathological correlates, was considered to have arisen incidentally and unrelated to test material toxicity.
No treatment-related blood chemical changes were detected in females treated with 600 and 250 mg/kg/day, or for animals of either sex treated with 50 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioural Assessment: Weekly open field arena observations did not reveal any treatment-related effects during the study. A higher number of males treated with 600 mg/kg/day showed urination compared with controls during the first week of the study, however, each animal only showed a single occasion of urination. In the absence of similar effects during later weekly assessments, or any other supporting evidence during the study, this observation was considered to be coincidental and unrelated to treatment. All remaining inter and intra groups variations in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.
Functional Performance Tests: No treatment-related effects were detected in the functional performance parameters investigated. Statistical analysis of the data did not reveal any significant inter group differences.
Sensory Reactivity Assessments: No treatment-related effects were detected in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be result of normal variation for arts of the strain and age used and were of no toxicological importance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There was one unscheduled death during the course of the study. One female treated with 250 mg/kg/day was killed in extremis on gestation Day 25, due to difficulties encountered during parturition. There were no histopathological changes in the tissues of this animal to directly suggest any reasons why parturition was problematical, although the animal did have only one functional kidney which may have been a contributory factor.
The following treatment-related changes were observed:
LIVER: Centrilobular hepatocyte enlargement was observed in relation to treatment for animals of either sex treated with 600 and 250 mg/kg/day. Two females treated with 50 mg/kg/day were similarly affected.
Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature.
THYROID GLAND: Follicular cell hypertrophy was observed as a consequence of treatment for males treated with 600 and probably also at 250 mg/kg/day, but not for 50 mg/kg/day males.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No adverse effect on mating performance, fertility or gestation lengths was detected.

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were demonstrated by offspring from all treatment groups.
The incidental findings observed in the control and treatment groups were those which are commonly observed in young offspring and considered unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Following birth, offspring survival in all groups was excellent and was unaffected by maternal treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Offspring bodyweight at Day 1 of age and subsequent bodyweight gain to Day 4 was unaffected by treatment, being essentially similar or slightly superior to concurrent control values. The statistically significant intergroup differences in litter weight observed were attributable to the previously discussed differences in litter size at the highest dose level.

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:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related macroscopic abnormalities were detected for the interim death or terminal kill offspring. The incidental findings consisting of the absence of milk in the stomach and small in size, are low incident findings in newly born rodent offspring, and unrelated to treatment.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The number of corpora lutea was similar in all groups, and was unaffected by treatment. The subsequent number of implantations at 600 mg/kg/day, however, was notably lower than controls, due to a higher incidence of pre-implantation loss.
At 250 mg/kg/day, mean pre-implantation loss were also slightly higher than controls. There was, however, notable intra-group variation and the subsequent mean number of implantations at this dose level and was not significantly lower than control values. Given the limitations of the small number of litters available for assessment, any association with treatment at this dose level was equivocal and differences were considered most likely to reflect normal biological variation. Preimplantation loss and the mean number of implantations at 50 mg/kg/day were essentially similar to controls.
Post-implantation loss was unaffected by treatment at all the treatment levels investigated and inter-group differences in mean litter size reflected the previously established pattern for implantations.
A lower number of offspring successfully demonstrated surface righting reflex at 600 mg/kg/day compared with offspring from the control group, on Day 1 post partum, despite the marginally superior Day 1 bodyweight at this dose level. Differences from control values were also observed at 250 and 50 mg/kg/day but, these were not as great as those at 600 mg/kg/day and were considered to reflect normal biological variation.
No adverse effect was detected for detachment of pinna. Offspring from the 250 mg/kg/day showed a statistically significant difference in completion of pinna detachment (pく0.05), however, when post coital ages were compared, there were no overt differences, and in the absence of a dose-related response, this intergroup difference was considered to have arisen incidentally.

Sex ratio was similar in all groups and did not indicate any selective effect of treatment on the survival of either sex.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: pre-implantation loss; surface righting reflex

Critical effects observed:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The study was conducted under GLP according to OECD guideline 422 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as reliable to assess the toxicity to reproduction (screening) in rats, repeated dose oral toxicity (short-term) is discussed in the respective chapter.
The effects observed in the liver is considered to be adaptive and not to represent an adverse health effect, as it is a common observation following the administration of any xenobiotic. The same applies for the responses in the thyroid, which are commonly associated with adaptive liver changes due to the induction of thyroxine metabolising enzymes. Additionally, the increase in adrenal weights observed at 600 mg/kg/day is a non-specific common 'stress response' in rodents, usually associated with increased steroidogenesis and an expansion of the adrenal cortex. The absence of pathological changes minimises the toxicological significance of this finding.
The most significant finding was a marked increase in pre-implantation loss at 600 mg/kg/day when compared to controls, leading to lower numbers of implantations and smaller litter size. Post-implantation and post-natal survival were not impaired. Additionally, a lower number of offspring successfully demonstrated surface righting reflex, compared with controls on Day 1 post partum, but in the absence of any effects on offspring growth or survival, the significance of this observation is unclear.

All animals, with the exception of one female receiving 600 mg/kg/day, mated within four days (at their first oestrus opportunity). The remaining female mated after fourteen days but failed to achieve pregnancy. In addition, two females treated with 250 mg/kg/day also failed to become pregnant. This is a low incidence finding in reproductive studies and in the absence of a convincing dose-related response, was considered to be of no toxicological significance.
The number of corpora lutea was similar in all groups, and was unaffected by treatment. The subsequent number of implantations at 600 mg/kg/day, however, was notably lower than controls, due to a higher incidence of pre-implantation loss. At 250 mg/kg/day, mean pre-implantation loss were also slightly higher than controls. There was, however, notable intra-group variation and the subsequent mean number of implantations at this dose level and was not significantly lower than control values. Given the limitations of the small number of litters available for assessment, any association with treatment at this dose level was equivocal and differences were considered most likely to reflect normal biological variation. Preimplantation loss and the mean number of implantations at 50 mg/kg/day were essentially similar to controls.
Post-implantation loss was unaffected by treatment at all the treatment levels investigated and inter-group differences in mean litter size reflected the previously established pattern for implantations. Following birth, offspring survival in all groups was excellent and was unaffected by maternal treatment. The results (Corpora Lutea, Implantation sites, pre- and post-implantation loss) are summarized in the following table:

Dose Level (mg/kg/day) Number of litters Number of Corpora Lutea Number of Implantation Sites Pre-Implantation Loss (%) Post-Implantation Loss (%)
0 (Control) 10 mean 16.3 13.6 15.7 4.0
sd 2.7 2.2 11.1 3.5
50 10 mean 15.2 12.2 18.5 6.5
sd 3.8 2.7 12.0 10.4
250 7 mean 15.5 11.7 25.8 9.2
sd 5.9 4.8 20.6 6.4
600 9 mean 15.6 10.3 32.9 7.3
sd 2.7 4.2 26.3 8.2

Despite the high intra-group variation of the mid-dose group, a dose-response is evident for the increase in pre-implantation loss. However, the standard deviation also increases from 11.1% up to a value of 26.3%.
Having a closer look at the Individual Litter Values (Implantation Losses and Survival Indices), it becomes evident that these high variances arise i.a. from two females showing pre-implantation losses of 82% and 58%, respectively. Additional two females showed pre-implantation losses of 42% and 43%, which also occurred in the control group (1 female with 42%). Given the small number of animals and the fact that high variations are present, these observations may as well be accidental. This is supported by the fact that pre-implantation losses were the only effects noted which are associated with reproductive toxicity. All other observations gave no indication of reproductive toxicity.
With regard to overall toxicity, it was reasonably concluded that all effects are non-adverse for the animal as such. Liver weights, both absolute and relative to terminal bodyweight were elevated for animals of either sex treated with 600 mg/kg/day, statistical significance was achieved for absolute weights for females. Centrilobular hepatocyte enlargement was observed in relation to treatment for animals of either sex treated with 600 and 250 mg/kg/day. Two females treated with 50 mg/kg/day were similarly affected. Although there is no reason to believe that the NOAEL of 600 mg/kg is not reasonable for the parental animals, those slight effects may have influenced the offspring, which would mean that effects of reproduction are only secondary ones. The increase in adrenal weights observed at 600 mg/kg/day is a non-specific common 'stress response' in rodents, making so stress evident, which certainly may have led to effects on the foetuses.
Regulation 1272/2008 states on reproductive toxicant that Category 1B is appropriate if available data provides clear evidence of an adverse effect on sexual function and fertility or on development in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of other toxic effects. However, when there is mechanistic information that raises doubt about the relevance of the effect for humans, classification in Category 2 may be more appropriate. The Regulation defines Hazard category 2 for reproductive toxicants as follows: „Suspected human reproductive toxicant Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification. Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects. Further the regulation says: Classification as a reproductive toxicant is intended to be used for substances which have an intrinsic, specific property to produce an adverse effect on reproduction and substances shall not be so classified if such an effect is produced solely as a non-specific secondary consequence of other toxic effects.
So the necessity to classify as substance as reproductive toxicant is only given if other, maternal effects were not present. This is not the case here, there were adaptive responses in the dams seen in all dose levels, and the adversity for the foetuses is unknown. Further, due to the high variations between the effects in the individual dose groups, evidence must be seen as not sufficiently convincing. Last but not least, taking into account the results from the available OECD 407, which only focuses, therefore in more detail, on adult animals, some overall systemic effects may have not been detected in this study, which may have led to the pre-implantation losses as the only effects noted on offspring. In this study, there were i.a. macroscopic and microscopic effects on the liver already at a dose level of 300 mg/kg, and the NOAEL of the OECD 407 study was found to be 100 mg/kg, related to degenerative changes in the liver. There it is concluded that there is not sufficient evidence to regard Styrenated diphenylamine as reproductive toxicant.
The observed LOAEL is with 600 mg/kg rather high, clearly above human relevance. There is no recommended limit value for classification as reproductive toxicant. However, e.g. the limit value for classification as STOT RE 2 with data from a subacute study is LOAEL = 300 mg/kg, the observed is LOAEL = 600 mg/kg is clearly above that value.

Summarizing, the authors of the OECD 422 report conclude that the oral administration of CAS No 68442-68-2 to rats by gavage, at dose levels of 600, 250 and 50 mg/kg/day, resulted in treatment-related effects at all dose levels. These effects however, were considered entirely adaptive in nature, therefore the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 600 mg/kg/day.
Treatment-related effects on reproduction were observed at 600 mg/kg/day. These were confined to an increase in pre-implantation losses, resulting in lower offspring numbers at this dose level. The NOAEL for reproductive toxicity was therefore considered to be 250 mg/kg/day, but it cannot be excluded that the observed effects were accidental or related to the adaptive responses in the dams. Hence, there is not sufficient evidence making a classification of Styrenated diphenylamine as reproductive toxicant Cat. 2 necessary.

Executive summary:

SUMMARY

Introduction. The study was designed to investigate the systemic toxicity and potential adverse effects on reproduction (including offspring development) of the test material and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996) under GLP.

Methods. The test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to fifty-four consecutive days, at dose levels of 50, 250 and 600 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (corn oil).

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected males and females from each dose group.

Pairing of animals within each dose group was undertaken on an one male: one female basis on Day 15 of the study, to produce litters.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of developmental landmarks.

Extensive functional observations were performed on five selected parental males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4post partum.

Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results.

Mortality. No treatment-related deaths were detected.

Clinical Observations. No clinically observable signs of toxicity were detected.

Behavioural Assessments. No treatment-related effects were detected.

Functional Performance Tests. No treatment-related effects were detected.

Sensory reactivity Assessments. No treatment-related effects were detected.

Bodyweights. No adverse effect on bodyweight was observed for males throughout the treatment period, or for females during the maturation, gestation or lactation phases of the study.

Food Consumption. No adverse effect on dietary intake was detected for males throughout the treatment period, or for females during the maturation, gestation or lactation phases of the study.

Water Consumption. No overt intergroup differences were detected.

Haematology. No treatment-related changes were detected prior to mating.

Blood Chemistry. Elevated alkaline phosphatase levels were detected for males treated with 600 mg/kg/day. Males treated with 600 and 250 mg/kg/day also showed reduced cholesterol levels. No such effects were detected for females treated with 600 or 250 mg/kg/day or for animals of either sex treated with 50 mg/kg/day.

 

Reproductive Screening:

Mating. No adverse effects on mating performance, fertility or gestation were detected.

Offspring Litter Size and Viability. Females treated with 600 mg/kg/day showed a higher percentage of pre-implantation losses in comparison to controls, resulting in the birth of less offspring per litter and lower total litter weights at this dose level. Mean offspring weights for treated animals were comparable to controls.

 

Offspring Development. Offspring from the 600 mg/kg/day dose group showed less successful completion of surface righting assessments. There were no treatment-related differences in pinna unfolding.

Litter Observations. There were no clinical signs to suggest an effect of treatment.

Pathology:

Necropsy.

Offspring: No treatment related macroscopic abnormalities were detected for the interim death offspring or for the remaining offspring at terminal kill.

Adults: No treatment-related macroscopic abnormalities were detected for the interim death female or for the remaining animals at terminal kill.

Organ Weights. Elevated liver and adrenal weights, both absolute and relative to terminal bodyweights, were detected for animals of either sex treated with 600 mg/kg/day.

Histopathology. Histopathological examination of adult tissue revealed the following treatment-related changes:

Liver: Centrilobular hepatocyte enlargement was observed for animals of either sex treated with 600 and 250 mg/g/day, with the effect extending into the female 50 mg/kg/day dose group.

Thyroid glands: Follicular cell hypertrophy was observed for males treated with 600 and probably also at 250 mg/kg/day. No such effects were detected for females at these dose levels, or for animals of either sex treated with 50 mg/kg/day.

Conclusion. The oral administration of CAS No 68442-68-2 to rats by gavage, at dose levels of 600, 250 and 50 mg/kg/day, resulted in treatment-related effects at all dose levels. These effects however, were considered entirely adaptive in nature, therefore the ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 600 mg/kg/day.

Treatment-related effects on reproduction were observed at 600 mg/kg/day. These were confined to an increase in pre-implantation losses, resulting in lower offspring numbers at this dose level. The NOAEL for reproductive toxicity was therefore considered to be 250 mg/kg/day. However, no classification as reproductive toxicant was considered necessary.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

There is an OECD 422 study (GLP) on the registered substance itself available, the tonnage-driven data requirements under REACH are fully met, so the database is of high quality.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

see effects on fertility, OECD 422 study, no effects on offspring development observed

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

There is an OECD 422 study (GLP) on the registered substance itself available, and although not very detailed, the tonnage-driven data requirements under REACH are fully met, so the database is of sufficient quality.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

A mode of action analysis in its classic sense cannot be performed for reproductive toxicity as there is only one reproductive toxicity screening study available in one species. However, the rat is an established model for human risk assessment, interspecies differences are well studied and allow transfer conclusions to humans for most chemicals exhibiting certain effects in the rat. Further, prolonged subacute (6-7 weeks) exposure is a sufficient time span to detect most systemic effects in rats; a carcinogenic MoAA which requires a longer duration can be reasonably excluded as the substance is not a directly acting mutagen, and in the present study there were no effects observed which may indicate tumour promoting effects. Further, exposure was sufficiently to cover relevant durations such as estrous cycles, pregnancy and a sufficient time of weaning. For details, please refer to the respective endpoint. Summarizing, the only effects noted which are associated with reproductive toxicity are pre-implantation losses in the high dose group. In this dose group, large inter-individual variations were noted, so due to the lack of other effects on reproductive toxicity this finding may be incidental. With regard to overall toxicity, it was reasonably concluded that all effects are non-adverse for the adult animal as such. Although there is no reason to believe that the NOAEL of 600 mg/kg is not reasonable for the parental animals, those slight effects may have influenced the offspring, which would mean that effects of reproduction are only secondary ones. There were effects noted in the liver, which were also considered adverse in the available OECD 407 study, and the increase in adrenal weights observed at 600 mg/kg/day is a non-specific common 'stress response' in rodents, making so stress evident, which certainly may also have led to effects on the foetuses. In the also available OECD 407 study, systemic adverse effects were already noted at 300 mg/kg bw/d. It is hence concluded that there is not sufficient evidence to regard Styrenated diphenylamine as reproductive toxicant. The observed LOAEL is with 600 mg/kg rather high, clearly above human relevance. However, there is no reason evident why these results in general cannot be transferred to humans.

Justification for classification or non-classification

Regulation 1272/2008 states on reproductive toxicant that Category 1B is appropriate if available data provides clear evidence of an adverse effect on sexual function and fertility or on development in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of other toxic effects. However, when there is mechanistic information that raises doubt about the relevance of the effect for humans, classification in Category 2 may be more appropriate. The Regulation defines Hazard category 2 for reproductive toxicants as follows: „Suspected human reproductive toxicant Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification. Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects. Further the regulation says: Classification as a reproductive toxicant is intended to be used for substances which have an intrinsic, specific property to produce an adverse effect on reproduction and substances shall not be so classified if such an effect is produced solely as a non-specific secondary consequence of other toxic effects.

So the necessity to classify as substance as reproductive toxicant is only given if other, maternal effects were not present. This is not the case here, there were adaptive responses in the dams seen in all dose levels, and the adversity for the foetuses is unknown. Further, due to the high variations between the effects in the individual dose groups, evidence must be seen as not sufficiently convincing. Last but not least, taking into account the results from the available OECD 407, which only focuses, therefore in more detail, on adult animals, some overall systemic effects may have not been detected in this study, which may have led to the pre-implantation losses as the only effects noted on offspring. In this study, there were i.a. macroscopic and microscopic effects on the liver already at a dose level of 300 mg/kg, and the NOAEL of the OECD 407 study was found to be 100 mg/kg. It is concluded that there is not sufficient evidence to regard Styrenated diphenylamine as reproductive toxicant.

The observed LOAEL is with 600 mg/kg rather high, clearly above human relevance. There is no recommended limit value for classification as reproductive toxicant. However, e.g. the limit value for classification as STOT RE 2 with data from a subacute study is LOAEL = 300 mg/kg, the observed is LOAEL = 600 mg/kg is clearly above that value.