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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The target substance maleic anhydride was tested in a multigeneration reproductive toxicity study (simila to OECD Guideline 416) with rats. no treatment-related effects on reproduction were observed with maleic anhydride at doses up to 55 mg/kg bw/day over two generations. Based on the results, the NOAEL for reproduction is considered to be 55 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
(proportion Male/female during mating 1/2; oestrus cycle and sperm parameters not analyzed)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Charles River CD rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The Charles River Breeding Laboratories, Inc ., Portage, Michigan
- Age at study initiation: (P/F0): 5-6 wks; (F1): 22 days
- Housing: single (besides mating and lactation period)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 16 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 25-60
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Maleic anhydride was admixed daily with the vehicle, Mazola corn oil using a tissue homogenizer.The test article was prepared at concentrations to permit administration at dosage levels of 20, 55 and 150 mg/kg/day at a constant dose volume of 10 ml/kg .
Details on mating procedure:
- M/F ratio per cage: 1/2
- Length of cohabitation: maximum of 15 days
- Proof of pregnancy: vaginal plug in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
P/F0: from study initiation to the end of the generation; F1: at 22 days of age and continued throughout the generation (Premating exposure period: F0 and F1, a minimum of 80 days) and throughout mating, gestation and lactation)
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
P/F0 rats were bred twice to produce the F1a and F1b litters. At weaning, ten males and 20 females from each dose group of the F1b litters were randomly selected to become parents of the F2a and F2b litters. A minimum of nine additional male and eight additional female pups per group were randomly selected to be used as replacements for animals dying prior to initiation of the generation.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
55 mg/kg bw/day (actual dose received)
Remarks:
Mid dose
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
10 males, 20 females
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (additionally, female body weights were measured at intervals during gestation and lactation)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OTHER:
Male and female fertility, mean gestation length
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [P/F0 and F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities (besides organ weights); possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- all P/F0 parents

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
These tissues were prepared for microscopic examination and weighed, respectively: adrenal, colon, heart, ileum, duodenum, kidney, liver, lung, bone marrow (sternum), brain, eye, mesenteric lymph node, mammary gland, pituitary, pancreas, salivary gland, skin, ovary, spleen, stomach, testis (with epididymis), thyroid (with a section of thrachea and esophagus), spinal cord (cervical), urinary bladder, prostate, uterus, all other gross lesions including tissue masses
Postmortem examinations (offspring):
SACRIFICE
- all F1 parents
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
These tissues were prepared for microscopic examination and weighed, respectively: adrenal, colon, heart, ileum, duodenum, kidney, liver, lung, bone marrow (sternum), brain, eye, mesenteric lymph node, mammary gland, pituitary, pancreas, salivary gland, skin, ovary, spleen, stomach, testis (with epididymis), thyroid (with a section of thrachea and esophagus), spinal cord (cervical), urinary bladder, prostate, uterus, all other gross lesions including tissue masses
Statistics:
All statistical analyses compared the treatment groups with the control group, with a level of significance at p<0 .05. Significance at p<0 .01 was also indicated. 1. Parental Body Weights: The parental body weights by sex were analysed by one-way analysis of variance, Bartlett's test for homogeneity of variances, and the appropriate t-test (for equal or unequal variances). Significant differences were determined using Dunnett's multiple comparison tables. Analyses for the P/F0 generation were conducted at one week prior to the F1a mating (week 11) and at termination of the generation (week 32). Analyses for the F1 generation were conducted at the first week of the generation (week 30), one week prior to the F2a mating (week 41) and at the termination of the generation (week 61). 2 . Male and Female Fertility: Male and female fertility indices were compared using the Chisquare test criterion with Yates' correction for 2 x 2 contingency tables and/or Fisher's exact probability test to judge levels of significant differences. 3. Pup Survival indices: The proportion of live pups at birth per total number born and the survival indices at lactation days 4, 7, 14 and 21 were compared by the Mann-Whitney U-test to judge significant differences. 4. Litter Size and Pup Body Weights: The mean number of liveborn pups per litter and mean body weights of pups were analysed by one-way analysis of variance, Barlett's test for homogeneity of variances, and the appropriate t- test (for equal or unequal variances). Significant differences were determined using Dunnett's multiple comparison tables. 5. Organ Weights: Absolute and relative organ weights (P/F0, F1, F2a and F2b) were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) using Dunnett's multiple comparison tables to judge significance of differences.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
For further details see section "Details on results" below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
Food consumption and compound intake (if feeding study):
no effects observed
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
CLINICAL SIGNS (PARENTAL ANIMALS): With the exception of a few cases of respiratory rales, the clinical appearance and behaviour of all treated animals were not remarkably different from the controls.

MORTALITY (PARENTAL ANIMALS): Mortality of adult males and females was 0 to 10% in the low and mid dose group and 65 to 70% in the high dose group.Intubation error was the cause of death for both low and mid dose rats and a single high dose male.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): P/F0 body weights in the high-dose group were significantly reduced by week 11 (males: -14.1%; females: -10.4%) in both sexes and this reduction increased as the generation progressed (males: -19.7% on study week 32; females: -18%). The mid-dose group means were low but not statistically significantly different from the controls, except at the end of the generation in the females. No effects in the low dose group in either sex during both generations. Mean maternal body weight gain during gestation and lactation during the Fla and Flb matings was unaffected by the administration of Maleic anhydride at all treatment levels tested.
No treatment-related effects on food consumption were observed in any group during the study (a slight decrease in mean female food consumption (both g/rat/day and g/kg/day) was observed throughout the P/F0 generation in the 20 mg/kg/day group, but this was considered as not treatment-related.

ORGAN WEIGHT (PARENTAL ANIMALS):
There were no obvious toxicologically significant variations in either mean absolute or relative (% body weight) organ weights for animals receiving 20, 55 or 150 mg/kg/day of maleic anhydride. There were statistically significant differences observed primarily for mean relative weights. The differences were contributed to the decrease in body weight gain and were not the result of a direct effect on the organs. There was also a statistically significant variation in the mean absolute and relative weight of the thyroid/parathyroid complex for females in the 20 mg/kg/day dosage group. there were no remarkable findings, however, microscopically. A biological significance is therefore doubtful.

GROSS PATHOLOGY (PARENTAL ANIMALS)/HISTOPATHOLOGY (PARENTAL ANIMALS):There were compound"related changes observed during postmortem examination of animals dying on study (DOS) or sacrificed at its termination (SAC). The changes included hydronephrosis/dilated pelvis, mottled kidneys/irregular surface and urinary calculi. Although, the incidences were small and randomly distributed among males and females (DOS/SAC) in the 20, 55 and 150 mg/kg bw/day dosage groups. Their toxicological significance was confirmed during microscopic examination. There was also a remarkaible incidence of gastric thickening, ulcerations, erosions and gaseous distension observed during macroscopic examination among animals in the 55 and 150 mg/kg bw/day dosage groups. A variety of gastritides were observed during microscopic examination. The primary cause(s), however, was not established. Thelesions were thereefore considered to be equivocally compound-related. There were other macroscopic changes observed in various organs. they were considered to be incidental and unrelated to the adminstration of maleic anhydride.
There were compound-related degenerative/inflammatory changes observed histologically in the kidney and urinary bladder of animals in the FO generation which died on study (DOS) or were sacrificed at the termination (SAC). In addition, there were inflammatory changes observed in the stomach of males and females (SAC/DOS) which had received 20, 55 or 150 mg/kg bw/day of maleic anhydrideThe changes were considered to be equivocally compound-related because their pathogenic origin was not clearly established.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Male and female fertility indices were reduced in the 20 and 150 mg/kg bw/day groups in the F1a mating when compared with the control group. The relation of these findings to treatment is doubtful since the F1b mating fertility indices were generally comparable at all treatment levels. No difficulties were observed at parturition among the treated and control females in either the F1a or F1b matings. There were no significant differences in the mean length of gestation between the 20, 55 and 150 mg/kg bw/day females and control group females in the F1a and F1b matings.
Key result
Dose descriptor:
NOAEL
Remarks:
(fertility)
Effect level:
55 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Dose descriptor:
LOAEL
Remarks:
(systemic)
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Dose descriptor:
LOEL
Remarks:
(local)
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: inflammatory changes in stomachs (it was not possible to conclude they were directly related to maleic anhydride)
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
For further details see section "Details on results" below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
Food consumption and compound intake (if feeding study):
no effects observed
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
CLINICAL SIGNS: Clinically, in both parental generations (P0/P1), respirator rales was observed with increased frequency among treated animals when compared to the control group. In addition, treated P1/F1 parental rats vigorously resisted the physical dosing procedure.

MORTALITY: Most of these deaths were attributed to gavage-related injuries . If these deaths are omitted, then a 0 to 10% mortality was observed in both the low and mid dose groups, and significant mortality was produced only in the high dose group (100% among high-dose females). As a result of poor survival, the high dose group was terminated in the F1 generation, and the study was reduced from a three generation to a two generation study

BODY WEIGHT:
At the 20 mg/kg/day level, no effect on mean male body weight was observed throughout the F1 generation (percentage difference -2 .6 on study week 61). The 55 mg/kg bw/day group exhibited moderate to slight decreases in mean male body weight which decreased in magnitude as the F1 generation progressed (i.e., percentage difference -10 and -5 .4 on study weeks 41 and 61, respectively). Marked reductions in mean male body weight were observed at the 150 mg/kg/day level (percentage difference -12 .9 on study week 41) prior to sacrifice of the remaining males.
Females in the 20 and 55 mg/kg bw/day groups showed no adverse effects/regarding mean body weight throughout the F1 generation ; values for these treated groups generally exceeded corresponding control group values . At the 150 mg/kg bw/day level, slight to moderate inhibition of mean female body weight was observed until study week 42 when mortality reached 100% . Percentage differences for these females ranged from -12 .5 to -6 .2% on weeks 30 and 35, respectively . No statistical significance was observed at any of the F1 points of analysis .
Mean maternal body weight gain during gestation in the 20 and 55 mg/kg bw/day groups was comparable to the control group during the F2 matings (F2a and F2b). The 20 mg/kg/day group exhibited mean maternal body weight gain in excess of the control group during lactation in the F2a mating and mean loss in maternal body weight during lactation in the F2a . mating . Slight reduction of mean maternal body weight gain and no gain in mean maternal body weight were observed during lactation in the F2a and F2b mating respectively, at the 55 mg/kg bw/day level.

ORGAN WEIGHT AND HISTOPATHOLOGY: In the F1 generation, kidney weights were significantly increased in females from the low and mid dose group ; however, there were no microscopic changes observed in these kidneys.

REPRODUCTIVE PERFORMANCE:
Male and female fertility indices for the 20 and 55 mg/kg bw/day groups for the F2a and F2b mating were comparable to those of the control group . In addition, these values were generally within the range of historical control values. It was noted that fertility in this study was reduced in all groups including the control when compared to control groups of previous studies. However, this generalized reduction is not considered an effect of treatment since the control group also had reduced fertility indices. The general reduction in fertility observed throughout the P0 and P1 generations and mortality contribute to a significant reduction in the available sample size of the data with regard to reproductive and litter parameters . In the P1 generation, 17 litters were available at the 55 mg/kg bw/daylevel with 100% mortality among the 150 mg/kg bw/day females by the time of the first F1 mating.
Findings with regards to parturition and length of gestation for the Fl generation were similar to the FO generation.No dystocia was observed of the F2a and F2b matings and the mean length of gestation of the treated females was comparable to the control group during both matings of the F1generation.
Key result
Dose descriptor:
NOAEL
Effect level:
55 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: Since 100% mortality was observed among female rats at 150 mg/kg bw/day.The high dose group was terminated in the F1 generation, and 55 mg/kg bw/day was the highest dose tested in the F1 generation.
Remarks:
The study was reduced from a three generation to a two generation study.
Key result
Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
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:
no effects observed
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
Histopathological findings:
no effects observed
Other effects:
no effects observed
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not examined
MORTALITY/VIABILITY (OFFSPRING):
- Pup viability (birth):There were no biologically meaningful or statistically significant differences between the treated and control rats in the mean numbers of viable or stillborn pups on lactation day 0 in the F1a and F1b litter.
- Pup survival to weaning:Treeatment with maleic anhydride at levels of 20, 55 or 150 mg/kg bw/day had no apparent adverse affect on pup survival to weaning in the F1a or F1b litters.

BODY WEIGHT: In the F1a litters, mean pup body weight at birth was comparable to the control group at the 20 and 55 mg/kg bw/day levels but significantly reduced (p<0.01) in the 150 mg/kg/day group. This effect was not observed in the F1b litters; mean pup body weight at birth in all treated groups of the F1b litters was comparable to the control groups.
Pup growth during lactation was unaffected by treatment with Maleic anhydride at dosage levels of 20 and 55 mg/kg bw/day in both mating of the P0 generation. However, at the 150 mg/kg/day level, inhibition of pup body weight was observed throughout lactation [with statistical significance on days 7, 14 and 21 (by sex),] in the F1a litters and after day 7 of lactation in the F1b litters.

ORGAN WEIGHTS (OFFSPRING): In the F1 generation, the absolute kidney weights of adult females in the low- and mid-dose groups were significantly increased to 108 and to 111%, respectively, of the control value.

GROSS PATHOLOGY (OFFSPRING)/HISTOPATHOLOGY (OFFSPRING): - F1: Low incidence of mineralization (2/12 males; 3/21 females) and nephrosis (2/12 males ; 5/21 females) in the kidney of animals receiving 150 mg/kg/day. There were no microscopic changes in these kidneys.

OTHER FINDINGS (OFFSPRING): No treatment-related effects were observed on indices of fertility for males and females in the F1 generation. No adverse effects on litter size and on pup survival were observed at doses up to 150 mg/kg bw/day in the F1 litters, or at 55 mg/kg bw/day in the F2 litters.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
55 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
sexual maturation
Remarks on result:
other: Since 100% mortality was observed among parental F1 female rats at 150 mg/kg bw/day. The high dose group was terminated in the F1 generation, and 55 mg/kg bw/day was the highest dose tested in the F1 generation.
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: increased absolute kidney weights of adult females.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For further details see section "Details on results" below
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:
no effects observed
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
MORTALITY/VIABILITY:
- Pup viability (birth): Very slight reductions in the mean number of viable pups at birth in the F2a litters were observed at the 20 and 55 mg/kg bw/day levels; there was no similar increase in the mean number of stillborn pups. These very slight reductions were not considered to represent a test article-related effect since values for both the 20 and 55 mg/kg bw/day groups (12.2 and 12.0, respectively) were comparable to the historical control value (12.3) and no similar decreases were noted in theF2b litters. The mean numbers of viable and stillborn pups at birth in the 20 and 55· mg/kg bw/day groups were comparable to the. control group in the F2b litters.
- Pup survival:There were no statistically significant differences in pup survival to weaning between treated and control litters in both matings of the FI generation at doses of 55 mg/kg/day and less.

BODY WEIGHT: Mean pup body weight at birth of treated litters in both matings of the Fl generation was statistically comparable to the control group. In the F2a litters, mean pup body weights In the low and intermediate dose groups were generally comparable to the control through lactation. However, in the F2b litters, slight to moderate reductions in mean pup body weight were observed in the 20 and 55 mg/kg/day groups throughout lactation. These reductions were statistically significant on days 14 (combined weights) and 21 (females only) of lactation in the 20 mg/kg/day group and days 4 before reduction (females only) and 21 (females only) of lactation at the 55 mg/kg/day level.
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
20 mg/kg bw/day
Treatment related:
no
Conclusions:
With reference to fertility, neither a dose-related reduction nor a pattern (during the two consecutive matings) within the parental (P0) generation suggested a treatment-related effect. In F1 generation 100% mortality was observed among female rats at 150 mg/kg bw/day. Therefore, the high dose group was terminated and 55 mg/kg bw/day was the highest dose tested in the F1 generation. No adverse effects on fertility were observed. Based on these observations the NOAEL(fertility) was derived at 55 mg/kg bw/d (highest dose tested under the conditions of this study).
Executive summary:

In a multigeneration generation reproduction study (similar to OECD Guideline 416) maleic anhydride (purity 99%) was administered to 10 male and 20 female rats/dose in by gavage at dose levels of 0, 20, 55 or 150 mg/kg bw/day). The rats were mated to produce two generations, each with two litters. Groups of the same size from the second litter were used for subsequent generations and were given the same dose of maleic anhydride as were their parents. Since 100% mortality was observed among parental F1 female rats at 150 mg/kg bw/day. The high dose group was terminated in the F1 generation and 55 mg/kg bw/day was the highest dose tested in the F1 generation. The study was reduced from a three generation to a two generation study..

Renal cortical necrosis occurred in high-dose P/F0 males and females. Increased kidney weights were observed in low- and mid-dose adult F1 females. Therefore, no NOAEL could be determined and the LOAEL (systemic) was regarded as 20 mg/kg bw/day. With reference to fertility, neither a dose-related reduction nor a pattern (during the two consecutive matings) within the parental (P0) generation suggested a treatment-related effect. In F1 generation 100% mortality was observed among female rats at 150 mg/kg bw/day. Therefore, the high dose group was terminated and 55 mg/kg bw/day was the highest dose tested in the F1 generation. No adverse effects on fertility was observed. Based on these observations the NOAEL(fertility) was derived at 55 mg/kg bw/d (highest dose tested under the conditions of this study).

This study is acceptable and satisfies the guideline requirement for a 2-generation reproductive study OECD 416 in rats.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
55 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
comparable to guideline study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a multigeneration generation reproduction study (similar to OECD Guideline 416) maleic anhydride (purity 99%) was administered to 10 male and 20 female rats/dose in by gavage at dose levels of 0, 20, 55 or 150 mg/kg bw/day). The ratswere mated to produce two generations, each with two litters. Groups of the same size from the second litter were used for subsequent generations and were given the same dose of maleic anhydride as were their parents.Since 100% mortality was observed among parental F1 female rats at 150 mg/kg bw/day. The high dose group was terminated in the F1 generation and 55 mg/kg bw/day was the highest dose tested in the F1 generation. The study was reduced from a three generation to a two generation study..

Renal cortical necrosis occurred in high-dose P/F0 males and females. Increased kidney weights were observed in low- and mid-dose adult F1 females. Therefore, no NOAEL could be determined and the LOAEL (systemic) was regarded as 20 mg/kg bw/day. With reference to fertility, neither a dose-related reduction nor a pattern (during the two consecutive matings) within the parental (P0) generation suggested a treatment-related effect. In F1 generation 100% mortality was observed among female rats at 150 mg/kg bw/day. Therefore, the high dose group was terminated and 55 mg/kg bw/day was the highest dose tested in the F1 generation. No adverse effects on fertility was observed. Based on these observations the NOAEL(fertility) was derived at 55 mg/kg bw/d (highest dose tested under the conditions of this study).This study is acceptable and satisfies the guideline requirement for a 2-generation reproductive study OECD 416 in rats.

Effects on developmental toxicity

Description of key information

The target substance maleic anhydride was tested in developmental toxicity study (similar to OECD Guideline 414) with female rats. No adverse effects were observed after oral administration. Therefore, based on the results, the NOAELs for both maternal toxicity and fetal toxicity of maleic anhydride in this study are considered to be higher or equal to 140 mg/kg bw/day. According to REACH Annex X, a developmental toxicity with a second specie (non-rodent) is required. However, since the target subtance maleic anhydride is manufacture under strictly control conditiions and maleic anhydride monomer complete polymerised with no contact with general population, relevant human exposure is excluded. Based on these considerations, no further testing is considered to be required for assessment of the developmental toxicity of maleic anhydride.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(administered volume in the control and high dose group is higher (1.4 ml/100 g bw) than the advised maximum volume in the guideline (0.4 ml/100 g bw))
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Charles River CD rats
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12 weeks
- Housing: individually housed, except during mating
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 10 days


Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Concentration in vehicle: 1% (w/v)
- Amount of vehicle (if gavage): 0.3-1.4 ml/100g bw
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from gestation day 6 through day 15
Frequency of treatment:
daily
Duration of test:
day 20 of gestation
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
low dose
Dose / conc.:
90 mg/kg bw/day (actual dose received)
Remarks:
mid dose
Dose / conc.:
140 mg/kg bw/day (actual dose received)
Remarks:
high dose
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 6, 9, 12, 15, and 20

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: fetal swellings, number of viable and nonviable fetuses
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [1/3 per litter ]
- Skeletal examinations: Yes: [2/3 per litter ]
Statistics:
All statistical analyses compared the treatment groups with the control group, with the level of significance at p<0.05. Male to female fetal sex ratio and number of litters with anomalies were compared using the Chi-square test criterion with Yates correction for 2 x 2 contingency tables and/or Fisher's exact probability test as described by Siegel to Judge significance of differences. The proportion of lite resorbed fetuses and postimplantation losses were compared by the Mann-Whitney U-test as described by Siegel and Weil to judge significance of differences. Mean number of corpora lutes, total implantations and viable fetuses were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie using Dunnett's multiple comparison table to judge significance of differences. Fetal body weights were compared by analysis of variance (hierarchal classification) and t-test as described by Steel and Torri using Dunnett's multiple comparison tables to judge significance of differences.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
For further details see section "Details on results" below.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For further details see section "Details on results" below.
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
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not examined
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Respiratory involvement and red nasal discharge were observed in all dosage groups. The incidence of these was higher in the treated groups, though not in a dose-related pattern. No treatment-related deaths (one rat in each dosage group died during the first part of treatment; the cause of death was not determined) nor abnormal behavior was observed in any of the maleic anhydride treated groups. Mean body weight gain was reduced in the 30 mg/kg/day dosage group for the first three days of treatment. There was a slight mean body weight loss in the 90 and 140 mg/kg/day dosage groups for the first three days of treatment. These reductions in weight gains resulted in reduced mean body weight gains over the entire treatment period in all treatment groups compared to the control (however, mean weight of all groups was within 5% of control on days 15 and 20. No biologically meaningful differences in the mean number of viable fetuses, implantations, post implantation losses, corpora lutea, or in the male to female sex ratio between any of the maleic anhydride treated groups and the control group. The general appearance and behavior of rats were not affected by treatment.
Dose descriptor:
NOAEL
Effect level:
>= 140 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Only slight effects on the body weight in the 90 and 140 mg/kg treatment groups. These effect were reversible, and there were no statistically significant effects on body weight at any of the times examined.
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For further details see section "Details on results" below.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
For further details see section "Details on resutls" below.
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mean fetal body weights were lower in the treatment groups than in the control group. This was not considered compound related due to the unusually high mean fetal body weight in this concurrent control group (mean: 4 g). External evaluation, internal examination, and skeletal observations of fetuses from all three treatment groups showed no anomalies in fetal development which could be attributed to maleic anhydride (slight increase in fetal malformations in the 30 (2/23 litters) and 140 mg/kg/day dosage group (3/21 litters) when compared to the control group (1/23 litters) is considered due to random occurrence due to the variety of abnomalities observed).
Dose descriptor:
NOAEL
Effect level:
>= 140 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
>= 140 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
In developmental toxicity study (equivalent to OECD 414) maleic anhydride (>99% purity) was administered to 25 female Wistar rats/dose in corn oil at dose levels of 0, 30, 90 or 140 mg/kg bw/day from days 6 through 15 of gestation. On day 20 of gestation the animals were sacrificed. Based on the results, the NOAEL for both maternal toxicity and fetal toxicity of maleic anhydride in this study is considered to be higher or equal to 140 mg/kg bw/day.
Executive summary:

In a developmental toxicity study (equivalent to OECD 414) maleic anhydride (>99% purtiy.) was administered to 25, rats/dose by gavage at dose levels of 0, 30, 90 or 140 mg/kg bw/day from days 6 through 15 of gestation.

There were no changes in appearance or behavior attributable to treatment with maleic Anhydride at dosage levels of 30, 90 and 140 mg/kg bw/day. Respiratory involvement and red nasal discharge were observed in all dosage groups. The incidence of these was higher in the treated groups, though not in a dose-related pattern. One rat in each dosage group dl.dd during the first part of treatment. The cause of death was not determined. There were reduced mean maternal body weight gains during the first three days of treatment in the 30 mg/kg bw/day dosage group when compared to the control group. In the 90 and 140 mg/kg bw/day dosage groups mean body weight losses were observed for the first 3 days of treatment. These reductions in weight gains resulted in reduced mean weight gains over the entire treatment period in all treatment groups when compared to the control group. There were no biologically meaningful differences in the mean number of viable fetuses, implantations, post implantation losses, corpora lutea or in the male to female sex ratio between any of the maleic anhydride treated groups and the control group. Based on the results, the NOAEL for maternal toxicity is considered to be higher than 140 mg/kg bw/day.

There were no treatment-related effects in developmental parameters. Therefore, based on the result the developmental NOAEL is also considered to be higher or equal to 140 mg/kg bw/day.

 

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
140 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
comparable to guideline study
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

In a developmental toxicity study according to OECD 414, groups of 25 pregnant female rats received 0, 30, 90 or 140 mg/kg test substance during gestation days 6-15. One adult female died in each experimental group. Dams in all experimental groups either failed to gain weight or lost weight between Days 6 and 9 of gestation; however, the effect was reversible and there were no statistically significant effects on body weight gains at any interval. Dams from all test groups produced normal-sized litters, with no evidence of post-implantation loss. Fetal body weights in all treatment groups were slightly reduced compared to controls but the reductions were statistically significant only in the 30 and 90 mg/kg groups. This is not considered to be compound-related because fetal weights for concurrent control and all treated groups were slightly greater than the historical control values. Malformations were observed in one fetus (1/23 litters) from the control group, two fetuses (2/23 litters) from the 30 mg/kg group and three fetuses (3/21 litters) from the 140 mg/kg group. There was no evidence of a dose-related increase in any specific malformation. Fetal variations were comparable both in type and frequency in the control and treated groups. There was, however, no maternal NOEL because females at 30 mg/kg failed to gain weight from gestation day 6-9 although this was not statistically significant. The NOAEL for both maternal and developmental toxicity was 140 mg/kg/day.

Justification for classification or non-classification

Based on the results, the target substance does not warrant classification for reproductive toxicity in accordance with CLP Regulation 1272/2008.

Additional information

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