Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The effects of succinic anhydride on reproduction can be assessed using data from a 2-generation reproduction study with a structurally similar chemical, maleic anhydride. No adverse effects on fertility were observed at doses up to 55 mg/kg/day over two generations. Sufficient information is available using this read-across approach and succinic anhydride would be expected to show similar effects.

Link to relevant study records
Reference
Endpoint:
multi-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline-comparable study published in a peer-reviewed journal
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
A two-generation reproductive toxicity study using maleic anhydride is available. Male and female rats were administered 0, 20, 55 or 150 mg/kg bw/day maleic anhydride in corn oil and were mated to produce two generations (Short et al., 1986). The high dose group (150 mg/kg bw/day) during the second generation was terminated due to treatment-related mortality. The lack of adverse findings in the reproductive toxicity study with maleic anhydride is also seen in carcinogenicity studies with succinic anhydride. No adverse findings in reproductive organs were observed in rats or mice in 2-year studies with succinic anhydride (NTP, 1990). No treatment-related effects on reproduction were observed with maleic anhydride at doses up to 55 mg/kg bw/day over two generations.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Weanling CD rats were acclimated to the laboratory for at least 10 days prior to study initiation. Rats were individually housed, except during mating and lactation, in wire mesh cages or plastic cages with corn-cob bedding. All animals were maintained in environmentally controlled rooms with 12-hr photoperiods and given free access to feed (Purina Rodent Chow) and water.
Route of administration:
oral: unspecified
Vehicle:
corn oil
Details on exposure:
Briquettes of maleic anhydride were finely ground with a mortar and pestle and suspended in corn oil with the aid of a tissue homogenizer. All doses were prepared daily in corn oil in order to minimize problems with stability. The concentration of maleic anhydride was varied so that the desired dose could be administered orally in a volume of 10 mL/kg bw.
Details on mating procedure:
Rats, in groups of 10 males and 20 females represented the F0 generation, and females were bred twice with males in the same dose group to produce F1a and F1b litters. Then 10 males and 20 females were randomly selected from the F1b litter to become parents of the F2a and F2b litters. During the mating period, each male was housed with two females for up to 15 days. The females were examined daily for evidence of mating as revealed by vaginal plugs or sperm-positive vaginal smears. The day evidence of copulation was observed was identified as Day 0 of gestation and the female was transferred to an individual plastic cage containing nesting material. Males were returned to their wire-mesh cages at the end of the mating period. Females for which no evidence of copulation was detected after the 15-day mating period were individually housed in plastic cages with bedding.

Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
Each generation was dosed a minimum of 80 days with maleic anhydride before its members were mated. Rats in groups of 10 males and 20 females were treated at 0, 20, 55 or 150 mg/kg bw/day (F0), Treatment began when the F0 rats were 5-6 weeks old, continued for at least 80 days prior to mating and continued to termination. For the F1 animals treatment began on Day 22 of age, continued for at least 80 days prior to mating and then continued to termination.
Frequency of treatment:
Daily
Details on study schedule:
Females in the F0 generation were bred twice with males in the same dose groups to produce F1a and F1b litters. Then 10 males and 20 females were randomly selected from the F1b litter to become parents of the F2a and F2b litters. Treatment began when F0 rats were 5-6 weeks and F1 animals were 22 days of age, and continued until the generation was terminated.
Remarks:
Doses / Concentrations: 0, 20, 55, or 150 mg/kg bw/day
Basis: nominal conc.
No. of animals per sex per dose:
10 males and 20 females/dose group
Control animals:
yes
Details on study design:
- Dose selection rationale: not specified
- Rationale for animal assignment (if not random): not specified
Positive control:
n.a.
Parental animals: Observations and examinations:
Rats were observed for signs of toxicity and body weights were recorded at intervals during the study.
Oestrous cyclicity (parental animals):
No details
Sperm parameters (parental animals):
No details
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, litters with more than 10 pups were reduced to five males and females.

PARAMETERS EXAMINED
The following parameters were examined in [F1, F2] offspring: body weight (day 0, day 4 and day 21 after birth)
Postmortem examinations (parental animals):
Histopathological evaluation was performed on approximately 30 tissues from each parent that died during the study, selected parents from all groups in the F0 generation, and selected parents from the control and mid-dose groups in the F1 generation.
Postmortem examinations (offspring):
Histopathological evaluation was performed on approximately 30 tissues from 10 pups/sex from the control and mid-dose groups from the F2b litters.
Statistics:
Several different statistical methods were used to compare measurements made on test animals to the corresponding values determined for controls. The methods and the measurements to which they were applied are analysis of variance and Dunnett's test for adult body weights, litter size and pup weights; Fisher's exact probability test for mortality and fertility data and Mann-Whitney U test for fetal body weights. In all instances, p< 0.05 was selected as the level of significance.
Reproductive indices:
Percentages for pregnant females and fertile males were calculated
Offspring viability indices:
Mean live pups/liiter on day 0, 4 and 21 after birth were recorded
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the F0 generation significant mortality occurred. For detailed information please refer to box "Details on results (P0)" and to table 1 in box " Any other information on results incl. tables".
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
In the F0 generation significant mortality occurred. For detailed information please refer to box "Details on results (P0)" and to table 1 in box "Any other information on results incl. tables".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Adult body weights were not affected in the low dose group. While there were some differences in mean body weights between control animals and those of the mid-dose group, none of the differences was statistically significant. In the high dose group, mean body weights of oth sexes of the F0 generation were significantly reduced by week 11, and this reduction persisted for the remainder of the test. For detailed information please refer to box "Details on results (P0)" and to table 1 in box "Any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
not specified
Water consumption and compound intake (if drinking water study):
not specified
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:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological changes noted for the F0 adults included a treatment-related effect on kidneys in the high dose group. Renal cortical necrosis in 60% of males and 15% of females was not observed in any other group.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Fertility was significantly reduced in the experimental groups at several times. However, there was neither a dose-related reduction nor a pattern within a generation that suggested the presence of a treatment-related effect. For detailed information please refer to table 2 in box " Any other information on results incl. tables"
With the exception of a few cases of respiratory rales, the clinical appearance and behaviour of F0 rats were not remarkably different from those of their controls. Respiratory rales also occurred in F1 rats and the incidence and severity appeared to increase with dose. These rats often vigorously resisted handling at the time of dosing. This behaviour may be related to irritating effects of maleic anhydride or a taste aversion to the test material. In the F0 generation, significant mortality occurred in adults of both sexes from the high dose group. The F1 generation had a greater number of deaths, many of which were attributed to gavage-related injuries. If these traumatic deaths are omitted, mortality in the F1 generation tended to parallel that of the F0 generation except for the increase recorded for low-dose males. Adult body weights were not affected in the low dose groups. While there were some differences in mean body weights in the mid-dose group, none of the differences were statistically significant. In the high dose group, mean body weights of both sexes of the F0 generation were significantly reduced by week 11 and this reduction persisted for the remainder of the study. The F1 generation showed a similar pattern except that the only significantly depressed mean body weight occurred in the high dose males at 30 weeks. Fertility was significantly reduced in the experimental groups at several times but there was no dose-related reduction or pattern within a generation that suggested treatment-related effects. Microscopic examination of tissues from F0 adults revealed compound-related changes in kidneys of rats from the high-dose group. Renal cortical necrosis was present in 60% of males and 15% of females from this group and was not observed in any other group. In the F1 generation, the absolute kidney weights of adult females in the low- and mid-dose groups were significantly increased to 108 and 111%, respectively, of control. There were no microscopic changes in these kidneys.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Respiratory rales occurred and increased with dose. In addition, the rats often vigorously resisted handling at the time of dosing.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
The F1 generation had a greater number of deaths, many of which were attributed to gavage-related injuries. If these traumatic deaths are omitted, mortality in the F1 generation tended to parallel that of the F0 generation except for the increase recorded for low-dose males. One animal in the latter group died of interstitial pneumonia. While no cause could be identified, the three other deaths in this group are not believed to be compound-related because no deaths among mid-dose males were attributed to to the test material and characteristic compound-indcued lesions discussed subsequently were not noted. For detailed information please refer to table 1 in box "Any other information on results incl. tables".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The F1 generation showed a pattern that was roughly similar to tzhe F0 generation,, except that the only significantly depressed mean body weight occurred in high-dose males at 30 weeks. For detailed information please refer to table 1 in box "Any other information on results incl. tables".
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):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the F1 generation, the absolute kidney weights of adult females in the low- and mid-dose groups were significantly increased to 108 and 111%, respectively, of the control value (2.31 g).
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In the F1 generation, the absolute kidney weights of adult females in the low- and mid-dose groups were significantly increased to 108 and 111%, respectively, of the control value (2.31 g). There were no microscopic changes in these kidneys.
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):
Fertility was significantly reduced in the experimental groups at several times. However, there was neither a dose-related reduction nor a pattern within a generation that suggested the presence of a treatment-related effect. For detailed results please refer to table 3 in box "Any other informationon results incl. tables".
Dose descriptor:
NOAEL
Effect level:
55 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Critical effects observed:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No effects on pup survival were noted at doses up to 150 mg/kg bw/day for the F1a and F1b litters.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A few incidental body weight changes were noted but no consistent effects that could be attributed to treatment with the test item.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not examined
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Treatment with maleic anhydride had no effect on litter size or on pup survival at doses up to 150 mg/kg/day in the F1a and F1b litters.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on litter size and on pup survival were observed with maleic anhydride at doses up to 150 mg/kg/day in F1 a and F1b litters
Critical effects observed:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The high-dose group was terminated during the second generation due to treatment-related mortality in adults.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Pup body weights were significantly reduced at a few of the observation periods. In the low- and mid-dose groups, pup weights were significantly reduced only at weaning of the F2b litters. However, these effects were observed only in one litter of a generation, and there was no pattern between litters of a generation or between generations that suggested the presence of a treatment-related effect. For detailed information please refer to table 4 in box " Any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The evaluation revealed no compound-related changes in organ weights.
Gross pathological findings:
not examined
Histopathological findings:
no effects observed
Description (incidence and severity):
The examination of tissues from revealed no compound-related changes in incidences of microscopic lesions.
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
55 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Reproductive effects observed:
not specified

Table 1. Mortality and body weight of adult rats treated with maleic anhydride during a multigeneration reproduction study

   

          Maleic anhydride (mg/kg/day)

 F0 generation    0 20 55 150 
 Males  Mortalitya  0 (0) 10 (10)  10 (0)  70b (60)b 
   Body weightc 143502700 142524697  143506666  143431b562b 
 Females  Mortalitya  0 (0) 0 (0)  5 (0)  65b (65)b 
   Body weightc  129289368 130281345  130280337  127259b317b 
 F1 generation          
 Males  Mortalitya  20 (0)  40 (40)b  40 (0)  75b (58)b
   Body weightd  113495722 107500703  103445683  85b431-- 
 Females  Mortalitya  20 (0) 30 (5)   52b (10)  100b (14)
   Body weightd  96265334 100272343  95265347  84247-- 
a Total dead/total treated x 100 (% mortality minus gavage-related deaths). Groups contained 10 to 12 males and 20 to 21 females; bSignificantly different from the appropriate control; cBody weight (g/rat) at weeks 0, 11 and 32 of study; dBody weights (g/rat) at weeks 30, 41 and 61 of study.  

Table 2. Fertility of rats treated with maleic anhydride during a multigeneration reproduction study

 

          Maleic anhydride (mg/kg/day)

   0 20 55 150 
 Females  F1a  14/20 (70)a 7/20 (35)b 14/20 (70) 7/20 (35)b
  F1b 10/20 (50) 8/2 (40) 11/19 (58) 6/10 (60)
 F2a  14/20 (70) 13/15 (87)  9/11 (82)  --
   F2b  12/16 (75) 12/14 (86) 8/10 (80) --
Males  F1a  8/10 (80)c 5/10 (50)  9/10 (90)  4/10 (40)b 
   F1b  6/10 (60)  5 /9 (56)  7/10 (70)  5/9 (56)
   F2a  9/10 (90) 6/6 (100) 6/6 (100) --
   F2b  8/8 (100) 7/7 (100)   5/5 (100)  --
a Number pregnant/number mated x 100 (% pregnant); bSignificantly different from control; c Number fertile/number mated x 100 (% fertile)

Table 3. Litter size of rats treated with maleic anhydride during a multigeneration reproduction study

              Maleic anhydride (mg/kg/day)
 Litter  Days after birth  0 20  55  150 
 F1a  0421 12.2a12.0 (9.9)b9.9  11.010.5 (9.3)9.3  11.611.2 (9.3)8.8  13.113.4 (10.0)10.0 
 F1b  0421 13.313.0 (9.8)9.8  10.39.6 (9.0)8.9  13.413.2 (9.9)9.8  11.310.8 (9.7)9.3 
 F2a  0421 13.413.1 (9.9)9.9 12.211.6 (10.0)9.9  12.011.8 (9.8)9.8  ------
 F2b  0421 10.510.4 (8.2)8.2  13.613.3 (9.8)9.7  14.013.8 (10.0)9.0  ------ 
aMean live pups/litter on indicated day. The number of pregnant females that gave birth on day 0 is presented in Table 2; bMean live pups/litter before litter reduction (mean live pups/litter after reduction to five pups/sex/litter when possible) 

Table 4. Body weight of pups from rats treated with maleic anhydride during a multigeneration reproduction study

            Maleic anhydride (mg/kg/day)
 Litter  Days after birth  0 20  55  150 
 F1a  0421 6.7a12 (11)c58 (56)  6.611 (11)54 (53)  6.712 (11)58 (55)  5.8b10 (9)46b(44)b
 F1b  0421 6.411 (10)53 (50)  7.1b12 (12)54 (54) 6.211 (10)51 (50)  6.310 (10)47 (46) 
 F2a  0421 6.611 (10)47 (45) 6.410 (10)47 (46) 6.710 (10)46 (45)  -- ----
 F2b  0421 6.811 (11)50 (57) 6.410 (9)45 (44)b 6.19 (9)43 (44)b ------ 
aMean weight (g) of both males and females; bSignificantly different from control; cMean weight (g) of males (mean weight (g) of females) 
Conclusions:
No treatment-related effects on reproduction were observed with maleic anhydride at doses up to 55 mg/kg/day over two generations. Adjustment for gavage related deaths is highlighted in the abstract, although mortality in the high dose F1 group can probably be attributed to a treatment effect, the similar effect in high dose F0 animals was affected by mal-administrations. Mortalities aside, there was no significant effect in terms of reduced pregnancy rates among females or fertility amongst males at doses up to 55 mg/kg bw/day. No adverse effects on fertility were apparent at 55 mg/kg bw/day administered over two generations. Maleic anhydride administered orally at 150 mg/kg bw/day was toxic to parental rats, resulting in mortality. No adverse effects on litter size or pup survival were noted in F1a or F1b litters from parents treated at up to 150 mg/kg bw/day but effects were seen in the 2a and F2b litters at this dose, while those in the 55 mg/kg bw/day regimen were unaffected. Therefore 55 mg/kg bw/day was indicated to be the highest NOAEL. The parental and offspring NOAEL in this study was 55 mg/kg bw/day.
Executive summary:

In a two-generation study similar to OECD guideline 416, CD rats (10 males and 20 females/group) received 0, 20, 55, or 150 mg/kg/day maleic anhydride in corn oil orally and were mated to produce two generations, each with two litters. Treatment-related mortality in the F0 and F1 generations occurred primarily in the high-dose group. There were some gavage-related deaths in the F0 generation and mortality was observed at lower doses in the F1 generation but these were not related to treatment. Renal cortical necrosis occurred in high dose F0 males and females. Increased kidney weights were observed in the low- and mid-dose adult F1 females. No significant reduction in the percentage of pregnant females or the percentage of fertile males was observed with maleic anhydride at doses up to 55 mg/kg/day over two generations. At 150 mg/kg/day, maleic anhydride was toxic to parental animals. No adverse effects on litter size or pup survival were observed at doses up to 150 mg/kg/day in F1a and F1b litters or 55 mg/kg/day in F2a and F2b litters. Based on the results, the NOAEL (maternal & reproductive) can be considered to be 55 mg/kg bw/day.

Cyclic anhydrides, such as maleic anhydride and succinic anhydride, share structural and physicochemical properties. Reproductive toxicity data on maleic anhydride can be used to read-across to succinic anhydride and similar results would be expected. A separate study investigating reproductive toxicity of succinic anhydride is not proposed and cannot be justified based upon animal welfare considerations.

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
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 study with maleic anhydride, a cyclic anhydride which shares structural and physicochemical properties with succinic anhydride, treatment-related mortality in the F0 and F1 generations occurred primarily at concentrations of 150 mg/kg/day.

Male and female rats were administered 0, 20, 55 or 150 mg/kg/day maleic anhydride in corn oil and were mated to produce two generations. The high dose group (150 mg/kg/day) during the second generation was terminated due to treatment-related mortality in adults. No treatment-related effects on reproduction were observed with maleic anhydride at doses up to 55 mg/kg/day over two generations.

Renal cortical necrosis was seen in F0 animals at this dose level. Increased kidney weights were observed in the low- and mid-dose adult F1 females. No significant reduction in the percentage of pregnant females or the percentage of fertile males was observed with maleic anhydride at doses up to 55 mg/kg/day over two generations. At 150 mg/kg/day, maleic anhydride was toxic to parental animals. No adverse effects on litter size or pup survival were observed at doses up to 150 mg/kg/day in F1a and F1b litters or 55 mg/kg/day in F2a and F2b litters.

The lack of adverse findings in the reproductive toxicity study with maleic anhydride is also seen in carcinogenicity studies with succinic anhydride. No adverse findings in reproductive organs were observed in rats or mice in 2-year studies with succinic anhydride (NTP, 1990). The two-generation reproductive toxicity study with maleic anhydride, an analogue of succinic anhydride, showed no adverse effects on reproduction. The available data are sufficient to show an absence of reproductive toxicity hazard for succinic anhydride and additional studies in animals cannot be justified either scientifically or in terms of animal welfare. The parental and offspring NOAEL was 55 mg/kg bw/day. 150 mg maleic acid/kg bw/day resulted in parental mortality and renal toxicity among the F1 generation. While the NOAEL value was derived from a study with maleic acid, it is considered to be a useful approximation to the succinic anhydride reproduction NOAEL, on the basis of physico-chemical/structural similarities.  

Effects on developmental toxicity

Description of key information
 The teratogenic effects of succinic anhydride have been investigated in three studies using pregnant CD-1 mice. These data provide sufficient information to determine that succinic anhydride induces a significant increase in malformations only at doses in the adult toxic range following intraperitoneal injection. In a developmental toxicity study conducted similar to OECD 414, the source substance maleic anhydride did not induce any effects at the high dose of 140 mg/kg bw/day. 
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Weanling rats were acclimated to the laboratory for at least 10 days prior to study initiation. Rats were individually housed, except during mating and lactation, in wire mesh cages or plastic cages with corn-cob bedding. All animals were maintained in environmentally controlled rooms with 12-hr photoperiods and given free access to feed (Purina Rodent Chow) and water.
Route of administration:
oral: unspecified
Vehicle:
corn oil
Details on exposure:
Briquettes of maleic anhydride were finely ground with a mortar and pestle and suspended in corn oil with the aid of a tissue homogenizer. All doses were prepared daily in corn oil in order to minimize problems with stability. The concentration of maleic anhydride was varied so that the desired dose could be administered orally in a volume of 10 mL/kg.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
Each generation was dosed a minimum of 80 days with maleic anhydride before its members were mated.Rats in groups of 10 males and 20 females were treated at 0, 20, 55 or 150 mg/kg bw/day (F0), Treatment began when the F0 rats were 5-6 weeks old, continued for at least 80 days prior to mating and continued to termination. For the F1 animals treatment began on Day 22, continued for at least 80 days prior to mating and then continued to termination
Frequency of treatment:
Daily
Duration of test:
Each generation was dosed a minimum of 80 days with maleic anhydride before its members were mated.Rats in groups of 10 males and 20 females were treated at 0, 20, 55 or 150 mg/kg bw/day (F0), Treatment began when the F0 rats were 5-6 weeks old, continued for at least 80 days prior to mating and continued to termination. For the F1 animals treatment began on Day 22, continued for at least 80 days prior to mating and then continued to termination
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
low dose
Dose / conc.:
90 mg/kg bw/day (nominal)
Remarks:
mid dose
Dose / conc.:
140 mg/kg bw/day (nominal)
Remarks:
high dose
No. of animals per sex per dose:
25 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
No further information
Maternal examinations:
no details
Ovaries and uterine content:
no details
Fetal examinations:
no details
Statistics:
no details
Indices:
no details
Historical control data:
no details
Details on maternal toxic effects:
The general appearance and behavior of rats were not altered by treatment. While one adult died in each of the experimental groups, the overall survival in these groups was 96%. Dams in the experimental groups either failed to gain weight or lost weight between Days 6 and 9 of gestation. However, this effect was reversible, and there were no statistically significant effects on body weight at any of the times examined.
Dose descriptor:
NOAEL
Effect level:
140 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Dams from all test groups produced normal-sized litters, and there was no evidence of postimplantation loss. Compared with concurrent controls, fetal body weights were slightly reduced for all test groups, but the reductions were statistically significant only in the low- and high-dose groups, However, this is not considered to be compound-related, because fetal weights for were slightly greater than the values for historical controls. Malformations were observed in one fetus (one litter) from the con-
trol group, two fetuses (two litters) from the low-dose group, and three fetuses (three litters) from the high-dose group. Since each malformation was a single occurrence and the malformations differed among the various groups, there was no evidence of a dose-related increase in any specific malformation. The fetal variations were comparable both in type and frequency in the control and treated groups.
Dose descriptor:
NOAEL
Effect level:
140 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects were observed
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In the teratology study, pregnant rats received 0, 30, 90, or 140 mg/kg/day maleic anhydride in corn oil orally from Days 6-15 of gestation and fetuses were examined for gross soft tissue and skeletal defects. Based on the result, the maternal and developmental NOAEL can be considered to be 140 mg/kg bw.
Executive summary:

In a developmental toxicity study (equivalent to OECD 414), CD rats (25 mated females/group) were treated orally with 30, 90, or 140 mg/kg/day of maleic anhydride from Day 6 through Day 15 of gestation. Mated females in the control group were treated in a similar manner with 14 mL/kg of corn oil. All females were sacrificed with carbon dioxide on Day 20 of gestation, and the fetuses were delivered by cesar-

ean section. All fetuses were weighed and examined for external abnormalities. Approximately one-third of the fetuses were placed in Bouin’s fixative and examined for soft tissue abnormalities (Wilson, 1965). The remaining fetuses were fixed in alcohol, cleared with potassium hy-droxide, stained with Alizarin Red S (Dawson, 1926), and examined for skeletal abnormalities.

Results showed, that the general appearance and behavior of rats were not altered by treatment. While one adult died in each of the experimental groups, the overall survival in these groups was 96%. Dams in the experimental groups either failed to gain weight or lost weight between Days 6 and 9 of gestation. However, this effect was reversible, and there were no statistically significant effects on body weight at any of the times examined.

Dams from all test groups produced normal-sized litters, and there was no evidence of postimplantation loss. Compared with concurrent controls, fetal body weights were slightly reduced for all test groups, but the reductions were statistically significant only in the low- and high-dose groups, However, this is not considered to be compound-related, because fetal weights for were slightly greater than the values for historical controls. Malformations were observed in one fetus (one litter) from the control group, two fetuses (two litters) from the low-dose group, and three fetuses (three litters) from the high-dose group. Since each malformation was a single occurrence and the malformations differed among the various groups, there was no evidence of a dose-related increase in any specific malformation. The fetal variations were comparable both in type and frequency in the control and treated groups.

Based on the results obtained, the maternal and developmental NOAEL can be considered to be 140 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
(before) 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline-comparable study published in a peer-reviewed journal
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: in vivo teratology screen
Principles of method if other than guideline:
A series of teratogenicity studies in mice were performed in the laboratory but only a subset of the data were presented to illustrate proposed methodology for estimating teratogenic potency (tD05) and Relative Teratogenic Index (RTI). Only the induction of major structural defects were evaluated. The intraperitoneal route of injection may not be the most appropriate for studying teratogenic effects in mice, since the irritant effects of succinic anhydride may induce artificial responses. Succinic anhydride is rapidly hydrolysed in blood (half-life of circa 2 minutes and 90% hydrolysis to succinic acid within 7 minutes), the hydrolysis product, succinic acid, is a substrate in the Kreb's cycle acting as a major metabolite in the citric acid cycle and ATP release endogenously within body cells. It is considered improbable that such an important endogenous metabolite/substrate would have any teratogenic properties.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
CD-1 mice were housed in climate-controlled conditions (20 °C, 50% humidity, 12 hr light cycle) with free access to water and NIH 31 chow.
Route of administration:
intraperitoneal
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
Dams were treated on 3 consecutive days (days 8-10) with single, daily, ip injections of 0.01 mL/g body weight. The highest dose level for teratology studies was normally within the 95% confidence limits of the calculated LD01 concentration, and a geometric progression of doses below this level was administered until no significant effects were observed. If minimal effects were observed at the LD01 dose, higher doses were utilized to ensure adequate assessment of teratogenic potential. For the majority of compounds, four or more dose levels were utilized, and in all cases there were at least 10 dams per group.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No details
Details on mating procedure:
Two female mice were housed with one male mouse overnight. Copulation was verified the following morning by the presence of a vaginal plug and this day was designated pregnancy day 1. To ensure animals used for testing were pregnant, only those animals which had gained 2 g body weight by day 8 were selected.
Duration of treatment / exposure:
3 days, pregnancy days 8-10
Frequency of treatment:
Daily
Duration of test:
18 days
Remarks:
The highest dose level was normally within the 95% confidence limits of the calculated LD01 concentration
No. of animals per sex per dose:
at least 10 dams/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: To estimate adult lethality, we administered each compound to female mice on 3 consecutive days as single, daily, intraperitoneal injections of 0.01 mL/gm body weight. A geometric progression of at least five doses was used, each of which utilized a group of 10 animals. Deaths were recorded for up to 14 days after the final injection.
Maternal examinations:
Not required in this determination of a method for assessing relative teratogenic index
Ovaries and uterine content:
Animals were sacrificed on day 18, the uteri were removed and the numbers and locations of live, dead and resorbing conceptuses were recorded.
Fetal examinations:
All live fetuses were examined for grossly observable malformations, weighed and then dissected with the aid of a stereomicroscope to evaluate visceral defects and to designate gender. Heads were fixed in Bouin's solution, sectioned by free-hand slicing and examined for abnormalities. Eviscerated fetuses were cleared and stained in KOH-Alizarin Red S prior to examination for skeletal abnormalities.
Statistics:
Chi-squared tests were used to determine goodness of fit of the observed data to the probit model. Probability values (P) are computed from standard distribution of X2 with k-2 degrees of freedom (k = number of doses used). When little heterogeneity was observed (p>0.010), confidence limits were computed using a "t" value of 1.98 (probability 0.05, infinity df). When probability from the X2 test was less than 0.10, the heterogeneity factor, h, was introduced. Variances and covariances were multiplied by this factor [h = X2/(k-2)], and confidence limits were computed using the "t" value at probability 0.05, k-2 df. This procedure has the effect of widening considerably the computed confidence limits when heterogeneity is observed. For RTI, computer-generated upper 95% confidence limits of LD01 and tD05 were taken to represent 2 SE above the predicted value and and an approximate variance of the LD01/tD05 ration was calculated. Tests for parallelism between the dose-response curves were performed by a computer program based upon the method of Finney.
Indices:
The dose-response trends of adult lethality and fetal malformations were examined by computerized probit analysis using an SAS program. Frequency of fetal malformation at each dose level was taken as the total number malformed/total number of live fetuses, and a constant background rate of malformation of 1.08% was included in the analysis. Thus, tD05, tD50, etc. refer to doses required to induce an additional 5% or 50% malformation rate above background rate. The Relative Teratogenic Index was calculated as LD01/tD05.
Historical control data:
The background rate of malformation was calculated from accumulated control data from the laboratory, where 19 malformed fetuses have been observed out of 1757 day 18, control fetuses. It should be noted that this analysis assumes that each individual fetus responds independently.
Mortality:
mortality observed, treatment-related
Description (incidence):
The LD50 was claculated to be 0.63 mmol/kg bw/day
Dose descriptor:
other: LD50
Effect level:
0.63 other: mmole/kg bw/day
Based on:
test mat.
Basis for effect level:
mortality
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: yes
Details on embryotoxic / teratogenic effects:Succinic anhydride only induces a significant incidence of malformations at doses within the adult lethality range.
From the best-fit lines, the doses required to induced specific incidences of malformation were computed as estimates of teratogenic potency. The median effective teratogenic dose, tD50, and the tD05 (dose required to induce 5% malformations) are presented in box effect levels and also in Table in box "Any other information on results incl. tables".
Dose descriptor:
other: tD50
Effect level:
79.02 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: teratogenicity
Dose descriptor:
other: tD05
Effect level:
31.02 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1. Computed adult lethal and teratogenic doses and relative teratogenic indices for succinic anhydride in the CD-1 mouse

   Adult lethality   mmol/kg/day (95% confidence limits)  Teratogenicity  mmol/kg/day (95% confidence limits)    Relative Teratogenic Index
 Compound  LD01 LD50  tD05  tD50   LD01/tD05 (95% confidence limits)
 Succinic anhydride  0.31 (0.15 - 0.40)  0.62 (0.52 - 0.70)  0.30 (--)a  0.79 (--)a  1.0 (--)
 Phthalic anhydride  0.37 (0.19 - 0.43) 0.51 (0.44 - 0.57)   0.40 (--)a  1.37 (0.61 )  0.91 (--)
a Heterogeneity factor included in calculation of confidence limits. Where confidence limits are not reported, they could not be calculated by computer program. 
Conclusions:
Succinic anhydride induces foetal malformations only at doses where significant adult lethality is induced. The route of exposure may have exaggerated the teratogenic potential artificially. The endogenous succinic acid/anhydride forms are improbable human teratogens.
Executive summary:

In a teratogenicity study, pregnant CD-1 mice were given intraperitoneal injections of succinic anhydride as a freshly prepared suspension of 0.5% carboxymethyl cellulose once per day on days 8 -10 of gestation. Only structural defects were included in the evaluation of teratogenic potential. For succinic anhydride, the median effective teratogenic dose, tD50, was 79 mg/kg/day (0.8 mmol/kg/day) and the tD05 was 31 mg/kg/day (0.3 mmol/kg/day). Succinic anhydride induced malformations in mice at doses nearly lethal to adults. This conclusion was based on a determination of a relative teratogenic index, defined as the ratio of minimum lethal dose to minimum teratogenic dose (LD01/tD05), equal to 1.0.

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
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 one teratogenicity study, pregnant CD-1 mice given ip injections of succinic anhydride on days 8 -10 of gestation showed malformations only at doses which were lethal to adults. Only structural defects were included in the evaluation of teratogenic potential and the median effective teratogenic dose, tD50, was 79 mg/kg/day and the minimum teratogenic dose, tD05, was 31 mg/kg/day (Fabro et al, 1982). As the effects occur together with maternal toxicity, the effects needs to be considered as secondary. The median teratogenic dose, tD50, for succinic anhydride was 0.8 mmol/kg/day and the tD05 was 0.3 mmol/kg/day. The relative teratogenic index, defined as the ratio of minimum lethal dose (LD01) to minimum teratagenic dose (tD05), was 1.0. The reliability of this study is limited because only major structural defects were included in the evaluation of teratogenic potency. Also, intraperitoneal injection is not a relevant route of human exposure. Additionally, this route is also not relevant for chemicals with low chemical stability in aqueous solutions because anticipated exposure by the oral, dermal or inhalation route will not result in the parent compound reaching the conceptus due to the rapid hydrolysis of succinic anhydride. This study can be scored as Klimisch category 2, reliable with restrictions.

In two other studies investigating the teratogenicity and chemical reactivity of selected anhydrides, ip injections of 50 mg/kg (Fabro et al, 1976) and 25 mg/kg (Brown et al, 1978) succinic anhydride given to CD-1 mice on gestation days 8 -10, or gestation days 11 -13, respectively, produced a significant increase in defects. Succinic anhydride was considered more potent than phthalic or maleic anhydride but was less active than propionic or acetic anhydride. These data suggest there may be a correlation between acylating ability and teratogenic potential. These are older studies were not conducted under GLP. There is limited information and only the abstracts have been published. A Klimisch score of 4, not assignable, may be appropriate.

The most reliable data is derived from a developmental toxicity study (equivalent to OECD 414) with the source substance maleic anhydride, in which CD rats (25 mated females/group) were treated orally with 30, 90, or 140 mg/kg/day of maleic anhydride from Day 6 through Day 15 of gestation. Mated females in the control group were treated in a similar manner with 14 mL/kg of corn oil. All females were sacrificed with carbon dioxide on Day 20 of gestation, and the fetuses were delivered by cesarean section. All fetuses were weighed and examined for external abnormalities. Approximately one-third of the fetuses were placed in Bouin’s fixative and examined for soft tissue abnormalities (Wilson, 1965). The remaining fetuses were fixed in alcohol, cleared with potassium hy-droxide, stained with Alizarin Red S (Dawson, 1926), and examined for skeletal abnormalities.

Results showed, that the general appearance and behavior of rats were not altered by treatment. While one adult died in each of the experimental groups, the overall survival in these groups was 96%. Dams in the experimental groups either failed to gain weight or lost weight between Days 6 and 9 of gestation. However, this effect was reversible, and there were no statistically significant effects on body weight at any of the times examined.

Dams from all test groups produced normal-sized litters, and there was no evidence of postimplantation loss. Compared with concurrent controls, fetal body weights were slightly reduced for all test groups, but the reductions were statistically significant only in the low- and high-dose groups, However, this is not considered to be compound-related, because fetal weights for were slightly greater than the values for historical controls. Malformations were observed in one fetus (one litter) from the control group, two fetuses (two litters) from the low-dose group, and three fetuses (three litters) from the high-dose group. Since each malformation was a single occurrence and the malformations differed among the various groups, there was no evidence of a dose-related increase in any specific malformation. The fetal variations were comparable both in type and frequency in the control and treated groups.

Based on the results obtained, the maternal and developmental NOAEL can be considered to be 140 mg/kg bw/day.

Justification for classification or non-classification

The available information on succinic anhydride suggests effects on fertility only at high concentrations and may be considered outside the criteria which lead to classification. Developmental toxicity appears as a secondary consequence of maternal toxicity such that classification may not be necessary. Futhermore, the developmental toxicity studies were conducted using intraperitoneal injection, which is not considered an appropriate route of administration which relates to potential human exposure. Based on existing data and the fact that the hydrolysis product, succinic acid, is ubiquitous in mammalian cells, classification of succinic anhydride as a reproductive toxicant is not triggered.