Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a screening study for reproduction/developmental toxicity study with rats, the NOAEL is considered to be 1000 mg/kg/day for reproductive performance of parents and for development of offspring.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 17,2010 to April 19,2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polynt Lot No. T208210033
- Manufacturing date: 11 Feb 2010
- Expiration date of the lot/batch: 11 Feb 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep container tightly closed. Keep in a dry, cool and well ventilated place.
- Storage at testing facility 5 years
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River (UK) Ltd.
- Choice:The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD (SD) strain was u sed because of the background data available in the laboratory.
- Age at study initiation: approximately 56 to 63 days of age
- Weight at study initiation: 290 to 330 g for males and 200 to 224 g for females
- Housing:barriered rodent facility (Building 30, Room 3021). The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. Before the study commenced the room was cleaned and disinfect ed. the rats are housed in P2000 or 2154 solid bottomed cages, or RB3 modified cages which were polypropylene cages with stainless steel grid flo oring. Wood based bedding was used when animals were housed in solid-bottomed cages, which was sterilised by autoclaving and changed at least twice a week.
- Diet : ad libitum , free access to a standard rodent diet (SDS VRF 1 Certified)
- Water : ad libitum, Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period:15 days
- Health check: twice daily during the acclimitisation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19 - 23 °C
- Humidity (%): 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12 cycle dark/light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: HHPA, was prepared for administration as a series of graded concentrations in the vehicle.

VEHICLE
- Amount of vehicle (if gavage):5 ml/kg
- Concentration in vehicle:0, 20, 60, 200 mg/ml
Details on mating procedure:
After a minimum of 15 days of treatment, F0 females were paired on a one-to—one basis with males from 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. Numbers of copulation plugs, an estimate of sperm density in the vaginal
smear and the quality of the spermatozoa were recorded and these records are retained with I5 the raw data for the study. The day on which mating was detected this day was designated Day 0 of gestation. Once mating had occurred, the males and females were separated and vaginal smearing 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:
Samples of each formulation from the first, fourth and last preparations were analysed for achieved concentration of the test substance. Four samples were taken from all groups. Two assays from each test group and one assay from the control group were analysed.
Duration of treatment / exposure:
The F0 animals were treated for 15 days before pairing, throughout pairing, gestation and lactation until the day before termination.F 0 males were terminated after approximately 48 days of treatment. F 0 females were allowed to litter and rear their offspring to Day 7 of age and were killed on Day 7 of lactation.
Frequency of treatment:
once a day during study
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:The dose levels of 0, 100, 300 and 1000 mg/kg/day were selected based on the results of a 7-day preliminary study
- Rationale for animal assignment: by numerical progression
- Negative control: the first group receive only a vehicle as negative control
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
A detailed physical examination was performed on the first day of treatment and weekly thereafter for F0 males until termination, and weekly for F0 females prior to pairing and on Days 0, 7, 14 and 20 after mating and Days l and 7 of lactation to monitor general health.

BODY WEIGHT: Yes
F0 males were weighed on the first day of treatment (Week 0) and weekly throughout treatment until termination. F 0 females were weighed on the first day of treatment and weekly until mating was detected. The females were weighed subsequently on Days 0, 6, 13 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION: Yes
Weekly from the start of treatment until the animals were paired for mating. For each F0 female, consumption was recorded for the periods Days 0-5, 6-12 and 13-19 after mating and Days 1-3 and 4-6 of lactation.


Oestrous cyclicity (parental animals):
For 15 days before pairing (including the day of pairing), daily vaginal smears (dry) were taken from all females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle.
Sperm parameters (parental animals):
Parameters examined in all P male parental generations:
testis weight, epididymis weight, sperm morphology of the seminiferous epithelium (staging of spermatogenic cycle)
Litter observations:
All litters were examined at approximately 24 hours after birth (Day l of age) and then daily thereafter.
Clinical signs: 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: mortality and consequent changes in litter size from Days 1-7 of age.
Sex ratio: Days l, 4 and 7 of age.
Bodyweight: Days l, 4 and 7 of age.
Postmortem examinations (parental animals):
All F0 animals were subject to a macroscopic examination of the tissues. All external features and orifices were examined visually.
After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. For F0 females, the numbers of corpora lutea on each ovary and the numbers of implantation sites in each uterine horn were counted.
Postmortem examinations (offspring):
For all F1 offspring, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. The necropsy also included an assessment for the presence of milk in the stomach, where this was possible.
Statistics:
For bodyweight, food consumption, organ weight, litter size and survival indices data a parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test. If the F1 approximate test for monotonicity of dose-response was not significant, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed.

For litter size and survival indices Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control.

For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate.

Sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect. Each treated group was compared to control using a Wald chi-square test.

For gestation length an exact two-tailed Linear-by-linear test, with equally spaced scores, was applied to all groups.

For oestrous cycles an exact one-tailed (upper-tail) Linear-by-linear test was applied to all groups.
Reproductive indices:
For males and females: % Mating, Conception rate (%), Fertility Index (%)
For females: Gestation index (%), Litter size
Offspring viability indices:
Post implantation survival index (%), Live birth index (%), Viability index (%), Lactation index (%).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
rales
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain observed during first week of treatment in animals treated at 1000 mg/kg bw/day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced food consumption observed during first week of treatment in animals treated at 1000 mg/kg bw/day
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Slightly higher spleen weight in animals treated at 1000 mg/kg bw/day
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY:
There were 7 mortalities. At 1000 mg/kg/day, during the two-week pre-pairing period 2 males and 2 females were killed for welfare reasons and 1 female was found dead. One further male was found dead at the end of Week 3. All of these animals showed noisy respiration (rales) prior to death or sacrifice and 1 male and 2 females showed gasping respiration. Two males and 2 females showed bodyweight loss prior to sacrifice. At 300 mg/kg/day, 1 female was killed for welfare reasons after showing noisy and gasping respiration and reduced body tone.
Among animals surviving to scheduled termination, signs of chin rubbing, salivation and noisy respiration (rales) were apparent after dosing of males and females at 1000 mg/kg/day. At 300 mg/kg/day, there was a low incidence of chin rubbing after dosing of both sexes and a low incidence of post-dose noisy respiration and salivation among females. Transient chin rubbing and/or post—dosing salivation is commonly observed where animals are dosed by oral gavage and the reaction is generally regarded as reflecting distaste of the dosing formulations rather than a sign of toxicity.
Clinical signs observed at routine physical examination which were considered to be related to treatment were restricted to noisy respiration (rales) among some animals receiving 1000 mg/kg/day.

BODY WEIGHT:
Males receiving 1000 mg/kg/day showed markedly low mean bodyweight gain during the first week of dosing compared with controls, while females receiving 1000 mg/kg/day showed mean bodyweight stasis during this period. Thereafter, the weight gain of males and females was essentially similar to controls to termination or pairing respectively, although, as a consequence of the performance during Week 1, overall gain for males at 1000 mg/kg/day was 80% that of controls. There was no clear effect of treatment at 300 or 100 mg/kg/day on mean bodyweight gain of males or females, and the mean bodyweight gain of females during gestation and lactation was unaffected by treatment at all dose levels investigated.

FOOD CONSUMPTION:
At 1000 mg/kg/day, males showed low mean food consumption during Week 1 and to a lesser extent during Week 2 of treatment, Females in this dose group also showed statistically significantly low food consumption during Week 1, however food intake in Week 2 was similar to control. There was no effect of treatment at 300 or 100 mg/kg/day on mean food consumption for males and females during the pre-pairing period. There was no effect of treatment on food consumption for females during gestation or lactation at any dose level investigated.

ORGAN WEIGHTS:
At 1000 mg/kg/day, mean absolute spleen weights were slightly high for males and females, and adjusted spleen weights were statistically significantly high for males. Mean absolute and adjusted ovary weights were marginally high and analysis of the adjusted weights attained statistical significance. There was no evidence of an effect of treatment on the limited list of weighed organs for males and females at 100 or 300 mg/kg/day.

GROSS PATHOLOGY:
Treatment-related macroscopic findings were restricted to stomach changes, apparent as a thickened glandular region (at 1000 mg/kg/day) and discoloured/roughened/thickened non glandular region (at 1000 mg/kg/day and to a much lesser extent at 300 mg/kg/day).The incidence and distribution of all other findings were consistent with the common background incidence.

MICROSCOPIC PATHOLOGY:
There were no changes in the reproductive organs which were considered related to treatment. The 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 abnormalities were observed.
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
CLINICAL SIGNS:
There were no clinical signs recorded among the offspring that were considered to be related to parental treatment with HHPA.

LITTER SIZE , SURVIVAL , INDICES AND RATIO:
There were no effects of parental treatment with HHPA at any dose level investigated on mean litter size or offspring survival to Day 7 of age.
The proportion of males per litter was 50% ± 8% males in all groups and was considered unaffected by parental treatment with HHPA. There were some minor intergroup differences in the percentage of males in each group; however, inspection of the individual litter values showed that most litters in each group contained 40-70% males and thus no effect of treatment was inferred.

BODYWEIGHT:
The mean birth weights of offspring in all treated groups, and subsequent weight gain to Day 7 of age was considered unaffected by parental treatment with HHPA at all dose levels investigated.

MACROPATHOLOGY:
Of the few offspring that died during the early post-natal period, most showed an absence of milk in the stomach as the predominant necropsy finding. This finding is common among offspring that die at an early age, and there were no findings observed which indicated a specific reason why these pups failed to thrive.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lack of treatment-related effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Based on the results of this reproduction/developmental toxicity screening study, it was concluded that a dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for reproduction/developmental toxicity in the CD rat.
Executive summary:

Reproductive and developmental toxicity has been investigated in a screening study conducted in accordance with OECD test methods. Groups of 10 males and 10 females received hexahydrophthalic Anhydride (HHPA) at dosages of 100, 300 and 1000 mg/kg/day for 15 days prior to pairing through to Day 6 after birth of the F1 generation.

During the study, data were recorded on clinical condition, bodyweight, food consumption, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken on the adult animals. The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed before macroscopic pathology investigations were undertaken at necropsy.

The results of these investigations lead to the conclusion that a dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for reproduction/developmental toxicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The effects hexahydrophthalic anhydride (HHPA) on fertility, pregnancy and early lactation of the offspring have been investigated in a reproduction/developmental toxicity screening study conducted according to OECD test methods. Groups of rats were dosed by oral gavage at levels of 100, 300 and 1000 mg/kg/day. Males were treated for 2 weeks prior to pairing and during pairing of all females until the day before necropsy, for a total of 6 weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods and for 7 days post partum. There were seven treatment-related mortalities during the course of the study, predominantly in the pre-pairing period, and attributed to aspiration of formulation droplets during the extubation of the dosing catheter causing local irritation to the airways, rather than evidence of systemic toxicity. Treatment-related signs were restricted to respiratory changes among males and females receiving 1000 mg/kg/day with a low incidence also observed among females at 300 mg/kg/day. Oestrous cycle length, mating performance, fertility, reproductive capacity and gestation length were unaffected by treatment. Bodyweight gain, survival and development of the offspring to Day 7 of age was unaffected by treatment. There were no treatment related macroscopic or microscopic abnormalities detected in the reproductive organs. Based on these findings it was concluded that 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for reproduction/developmental toxicity in the rat.This study is acceptable and satisfies the guideline requirement for a reproduction and developmental toxicity screening study in rats (OECD 421).

 

A "higher tier" study such as a 2 -generation study was waived for the following reasons:

The substance is classified as both a skin and respiratory sensitiser in accordance with the requirements of Directive 67/548/EEC (DSD) and Regulation 1272/2008 (CLP). As such, sensitisation is to be regarded as the most sensitive end-point for which no DNEL can be derived due to the lack of dose-response data. Instead, a qualitative approach must be applied to assess and control the risks (in accordance with "Guidance on information requirements and chemical safety assessment, Chapter R8: Characterisation of dose (concentration) - response for human health"). Under consideration of these aspects, together with the available information of the OECD 421 study, further testing would not be in line with current concerns regarding animal welfare and the use of animals in scientific experiments.




Effects on developmental toxicity

Description of key information

In a screening study for reproduction/developmental toxicity study with rats, the NOAEL is considered to be 1000 mg/kg/day for reproductive performance of parents and for development of offspring.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2010-02-17 to 2010-04-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 421
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polynt Lot No. T208210033
- Manufacturing date: 11 Feb 2010
- Expiration date of the lot/batch: 11 Feb 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep container tightly closed. Keep in a dry, cool and well ventilated place.
- Storage at testing facility 5 years
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River (UK) Ltd.
- Choice:The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD (SD) strain was used because of the background data available in the laboratory.
- Age at study initiation: approximately 56 to 63 days of age
- Weight at study initiation: 290 to 330 g for males and 200 to 224 g for females
- Housing:barriered rodent facility (Building 30, Room 3021). The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. Before the study commenced the room was cleaned and disinfected. the rats are housed in P2000 or 2154 solid bottomed cages, or RB3 modified cages which were polypropylene cages with stainless steel grid flo oring. Wood based bedding was used when animals were housed in solid-bottomed cages, which was sterilised by autoclaving and changed at least twice a week.
- Diet : ad libitum , free access to a standard rodent diet (SDS VRF 1 Certified)
- Water : ad libitum, Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period:15 days
- Health check: twice daily during the acclimitisation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19 - 23 °C
- Humidity (%): 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12 cycle dark/light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: HHPA, was prepared for administration as a series of graded concentrations in the vehicle.

VEHICLE
- Amount of vehicle (if gavage):5 ml/kg
- Concentration in vehicle:0, 20, 60, 200 mg/ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation from the first, fourth and last preparations were analysed for achieved concentration of the test substance. Four samples were taken from all groups. Two assays from each test group and one assay from the control group were analysed.
Details on mating procedure:
After a minimum of 15 days of treatment, F0 females were paired on a one-to—one basis with males from 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. Numbers of copulation plugs, an estimate of sperm density in the vaginal
smear and the quality of the spermatozoa were recorded and these records are retained with I5 the raw data for the study. The day on which mating was detected this day was designated Day 0 of gestation. Once mating had occurred, the males and females were separated and vaginal smearing discontinued. The pre-coital interval was calculated for each female as the time elapsing between initial pairing and detection of mating.
Duration of treatment / exposure:
The F0 animals were treated for 15 days before pairing, throughout pairing, gestation and lactation until the day before termination.F 0 males were terminated after approximately 48 days of treatment. F 0 females were allowed to litter and rear their offspring to Day 7 of age and were killed on Day 7 of lactation.
Frequency of treatment:
Once a day during study
Duration of test:
The F0 animals were treated for 15 days before pairing, throughout pairing, gestation and lactation until the day before termination.F 0 males were terminated after approximately 48 days of treatment. F 0 females were allowed to litter and rear their offspring to Day 7 of age and were killed on Day 7 of lactation.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:The dose levels of 0, 100, 300 and 1000 mg/kg/day were selected based on the results of a 7-day preliminary study
- Rationale for animal assignment: by numerical progression
- Negative control: the first group receive only a vehicle as negative control
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
A detailed physical examination was performed on the first day of treatment and weekly thereafter for F0 males until termination, and weekly for F0 females prior to pairing and on Days 0, 7, 14 and 20 after mating and Days l and 7 of lactation to monitor general health.

BODY WEIGHT: Yes
F0 males were weighed on the first day of treatment (Week 0) and weekly throughout treatment until termination. F 0 females were weighed on the first day of treatment and weekly until mating was detected. The females were weighed subsequently on Days 0, 6, 13 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION: Yes
Weekly from the start of treatment until the animals were paired for mating. For each F0 female, consumption was recorded for the periods Days 0-5, 6-12 and 13-19 after mating and Days 1-3 and 4-6 of lactation.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation Day 7
- Organs examined: Examination of external appearance, appearance of the tissues and organs examined after opening of the cranial, thoracic and abdominal cavities. Abnormalities recorded with details of location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
- Organ weights: Males - epididymides, spleen, testes; Females - Ovaries, spleen
- Tissues fixed and preserved: (from all animals) - Abnormalities, Adrenal glands, Brain, Caecum, Colon, Duodenum, Epididymides, Eyes, Femur, Head, Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes (mandibular, mesenteric and left axillary), Mammary area (caudal and cranial), Ovaries, Pituitary gland, Prostate gland, Rectum, Sciatic nerve, Seminal vesicles with coagulating gland, Skeletal muscle, Spinal cord, Spleen, Sternum with marrow, Stomach, Testes, Thymus, Thyroid with parathyroids, Trachea, Urinary bladder, Uterus – cervix, Vagina

HISTOPATHOLOGY: Yes
Tissues examined - Ovaries, testes and epididymides (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) of the animals in the control and high dose groups..
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
SACRIFICE
- The F1 offspring were sacrificed at 7 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes
- Skeletal examinations: No
- Head examinations: Yes
For all F1 offspring, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. The necropsy also included an assessment for the presence of milk in the stomach, where this was possible.
Statistics:
For bodyweight, food consumption, organ weight, litter size and survival indices data a parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test. If the F1 approximate test for monotonicity of dose-response was not significant, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed.

For litter size and survival indices Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control.

For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate.

Sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect. Each treated group was compared to control using a Wald chi-square test.

For gestation length an exact two-tailed Linear-by-linear test, with equally spaced scores, was applied to all groups.

For oestrous cycles an exact one-tailed (upper-tail) Linear-by-linear test was applied to all groups.
Indices:
Gestation index, pre-implantation mortality, post-implantation mortality, intra-uterine mortality, post-natal mortality, sex ratio, survival index
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Rales
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain observed during first week of treatment in animals treated at 1000 mg/kg bw/day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced food consumption observed during first week of treatment in animals treated at 1000 mg/kg bw/day
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Slightly higher spleen weight in animals treated at 1000 mg/kg bw/day
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY:
There were 7 mortalities. At 1000 mg/kg/day, during the two-week pre-pairing period 2 males and 2 females were killed for welfare reasons and 1 female was found dead. One further male was found dead at the end of Week 3. All of these animals showed noisy respiration (rales) prior to death or sacrifice and 1 male and 2 females showed gasping respiration. Two males and 2 females showed bodyweight loss prior to sacrifice. At 300 mg/kg/day, 1 female was killed for welfare reasons after showing noisy and gasping respiration and reduced body tone.
Among animals surviving to scheduled termination, signs of chin rubbing, salivation and noisy respiration (rales) were apparent after dosing of males and females at 1000 mg/kg/day. At 300 mg/kg/day, there was a low incidence of chin rubbing after dosing of both sexes and a low incidence of post-dose noisy respiration and salivation among females. Transient chin rubbing and/or post—dosing salivation is commonly observed where animals are dosed by oral gavage and the reaction is generally regarded as reflecting distaste of the dosing formulations rather than a sign of toxicity.
Clinical signs observed at routine physical examination which were considered to be related to treatment were restricted to noisy respiration (rales) among some animals receiving 1000 mg/kg/day.

BODY WEIGHT:
Males receiving 1000 mg/kg/day showed markedly low mean bodyweight gain during the first week of dosing compared with controls, while females receiving 1000 mg/kg/day showed mean bodyweight stasis during this period. Thereafter, the weight gain of males and females was essentially similar to controls to termination or pairing respectively, although, as a consequence of the performance during Week 1, overall gain for males at 1000 mg/kg/day was 80% that of controls. There was no clear effect of treatment at 300 or 100 mg/kg/day on mean bodyweight gain of males or females, and the mean bodyweight gain of females during gestation and lactation was unaffected by treatment at all dose levels investigated.

FOOD CONSUMPTION:
At 1000 mg/kg/day, males showed low mean food consumption during Week 1 and to a lesser extent during Week 2 of treatment, Females in this dose group also showed statistically significantly low food consumption during Week 1, however food intake in Week 2 was similar to control. There was no effect of treatment at 300 or 100 mg/kg/day on mean food consumption for males and females during the pre-pairing period. There was no effect of treatment on food consumption for females during gestation or lactation at any dose level investigated.

ORGAN WEIGHTS:
At 1000 mg/kg/day, mean absolute spleen weights were slightly high for males and females, and adjusted spleen weights were statistically significantly high for males. Mean absolute and adjusted ovary weights were marginally high and analysis of the adjusted weights attained statistical significance. There was no evidence of an effect of treatment on the limited list of weighed organs for males and females at 100 or 300 mg/kg/day.

GROSS PATHOLOGY:
Treatment-related macroscopic findings were restricted to stomach changes, apparent as a thickened glandular region (at 1000 mg/kg/day) and discoloured/roughened/thickened non glandular region (at 1000 mg/kg/day and to a much lesser extent at 300 mg/kg/day).The incidence and distribution of all other findings were consistent with the common background incidence.

MICROSCOPIC PATHOLOGY:
There were no changes in the reproductive organs which were considered related to treatment. The 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 abnormalities were observed.
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lack of treatment-related effects
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
no
Conclusions:
In a screening study of reproductive toxicity conducted in accordance with OECD test methods, the substance caused no changes in male and female reproductive performance (gonad function, mating behaviour, conception, pregnancy, parturition) and dam’s delivery data. There were no test item related effects on offspring’s development. Based on these observations the No Observed (Adverse) Effect Level for reproductive performance of the male and female rats was regarded as 1000 mg/kg body weight/day and the NO(A)EL for F1 Offspring was 1000 mg/kg body weight/day
Executive summary:

In a screening study of reproductive toxicity conducted in accordance with OECD test methods, the substance caused no changes in male and female reproductive performance (gonad function, mating behaviour, conception, pregnancy, parturition) and dam’s delivery data. There were no test item related effects on offspring’s development. Based on these observations the No Observed (Adverse) Effect Level for reproductive performance of the male and female rats was regarded as 1000 mg/kg body weight/day and the NO(A)EL for F1 Offspring was 1000 mg/kg body weight/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The effects hexahydrophthalic anhydride (HHPA) on fertility, pregnancy and early lactation of the offspring have been investigated in a reproduction/developmental toxicity screening study conducted according to OECD test methods. Groups of rats were dosed by oral gavage at levels of 100, 300 and 1000 mg/kg/day. Males were treated for 2 weeks prior to pairing and during pairing of all females until the day before necropsy, for a total of 6 weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods and for 7 days post partum. There were seven treatment-related mortalities during the course of the study, predominantly in the pre-pairing period, and attributed to aspiration of formulation droplets during the extubation of the dosing catheter causing local irritation to the airways, rather than evidence of systemic toxicity. Treatment-related signs were restricted to respiratory changes among males and females receiving 1000 mg/kg/day with a low incidence also observed among females at 300 mg/kg/day. Oestrous cycle length, mating performance, fertility, reproductive capacity and gestation length were unaffected by treatment. Bodyweight gain, survival and development of the offspring to Day 7 of age was unaffected by treatment. There were no treatment related macroscopic or microscopic abnormalities detected in the reproductive organs. Based on these findings it was concluded that 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for reproduction/developmental toxicity in the rat.This study is acceptable and satisfies the guideline requirement for a reproduction and developmental toxicity screening study in rats (OECD 421).

 

A "higher tier" study such as a formal developmental toxicity study was waived for the following reasons:

The substance is classified as both a skin and respiratory sensitiser in accordance with the requirements of Directive 67/548/EEC (DSD) and Regulation 1272/2008 (CLP). As such, sensitisation is to be regarded as the most sensitive end-point for which no DNEL can be derived due to the lack of dose-response data. Instead, a qualitative approach must be applied to assess and control the risks (in accordance with "Guidance on information requirements and chemical safety assessment, Chapter R8: Characterisation of dose (concentration) - response for human health"). Under consideration of these aspects, together with the available information of the OECD 421 study, further testing would not be in line with current concerns regarding animal welfare and the use of animals in scientific experiments.

Justification for classification or non-classification

Classification is not justified based on the available information from a screening study for reproduction/developmental toxicity.