Dioctyl maleate, branched

Toxicity to reproduction

Administrative data

Endpoint:

multi-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1986

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study has been disregarded due to significant methodological difficiencies compared to the OECD 416 guideline. • The administered dose was greater than that recommended for corn oil suspensions (10 ml/kg bw vs 4 ml/kg bw) • The parental (P0) animals were older than recommended (12 week vs 5-9 weeks) at the start of dosing • There was a high occurance of gavage related unscheduled fatalities in the F1 generation. Number of deaths from gavage is not adequately reported. • The test material is corrosive to the skin, however no accomodation or variation was made for this in dose preparation. • The highest dose group was subject to unscheduled termination at the F1 generation due to higher than expected toxicity, therfore no conclusion can be reached for the F2 generation.

Justification for type of information:

Study conducted outside the EU over 10 years before the EU cosmetic testing ban came into effect.

Data source

Reference

Reference Type:

publication

Title:

Teratology and Multigeneration Reproduction Studies with Maleic Anhydride in Rats

Author:

Short, R.D., Johannsen, F.R., Levinskas, G.J., Rodwell, D.E. & Schardein, J.L.

Year:

1986

Bibliographic source:

FUNDAMENTAL AND APPLIED TOXICOLOGY, Vol 7, pp. 359-366

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Principles of method if other than guideline:

The study has been disregarded due to significant methodological difficiencies compared to the OECD 416 guideline.
• The administered dose was greater than that recommended for corn oil suspensions (10 ml/kg bw vs 4 ml/kg bw)
• The parental (P0) animals were older than recommended (12 week vs 5-9 weeks) at the start of dosing
• There was a high occurance of gavage related unscheduled fatalities in the F1 generation. Number of deaths from gavage is not adequately reported.
• The test material is corrosive to the skin, however no accomodation or variation was made for this in dose preparation.
• The highest dose group was subject to unscheduled termination at the F1 generation due to higher than expected toxicity, therfore no conclusion can be reached for the F2 generation.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Maleic anhydride

EC Number:

203-571-6

EC Name:

Maleic anhydride

Cas Number:

108-31-6

Molecular formula:

C4H2O3

IUPAC Name:

furan-2,5-dione

Test material form:

not specified

Specific details on test material used for the study:

Maleic anhydride was supplied by Monsanto Company as white briquettes with a purity of greater than 99%

Test animals

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Age at study initiation: 12 weeks
- Weight at study initiation: F 242-244 g
- Fasting period before study: No
- Housing: Wire mesh cages or plastic cages with corn-cob bedding
- Diet (e.g. ad libitum): Purina Rodent Chow, Ralston-Purina, St. Louis, Mo. - Ad libitum
- Water (e.g. ad libitum): - Ad libitum
- Acclimation period: 10 days

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Maleic anhydride was suspended in corn oil as described previously; however, the concentration was varied so that the desired dose could be administered orally in a volume of 10 ml/kg.

Details on mating procedure:

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 specified.

Duration of treatment / exposure:

Not specified.

Frequency of treatment:

Daily

Details on study schedule:

These rats represented the P0 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. Each generation was dosed a minimum of 80 days with maleic anhydride before its members were mated. Treatment began when P0 rats were 5 to 6 weeks and F1 animals were 22 days of age, and continued until the generation was terminated.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

55 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males / 20 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose selection was based upon the teratogenicity study reported in section 7.8.2 (Short et al, 1986), and was reported in the sample publication.

Positive control:

Not specified.

Examinations

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):

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 F1 / F2 offspring:
• number and sex of pups,
• stillbirths,
• live births,
• postnatal mortality,
• presence of gross anomalies,
• weight gain,
• physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

A histopathological evaluation was performed on approximately 30 tissues from each parent that died during the study, selected parents from all groups in the P0 generation, selected parents from the control and mid-dose groups in the Fl generation, and 10 pups/sex from the control and mid-dose groups from the F2b litters.

Postmortem examinations (offspring):

A histopathological evaluation was performed on approximately 30 tissues from each parent that died during the study, selected parents from all groups in the P0 generation, selected parents from the control and mid-dose groups in the Fl generation, and 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 analyis of variance and Dunnett's test (Steel and Torrie, 1960) for adult body weights, litter size, and pup body weights; Fisher's exact probability test (Siegel, 1956) for mortality and fertility data; Mann-Whitney U test (Siegel, 1956) for fetal body weights; and x2 test with Yates' correction or Fisher's exact probability test (Siegel, 1956) for litters with anomalies. In all instances, p < 0.05 was selected as the level of significance.

Reproductive indices:

yes, see Statistics.

Offspring viability indices:

yes, see Statistics.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

With the exception of a few cases of respiratory rales, the clinical appearance and behavior of these Fo rats were not remarkably different from those of their controls.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the P0 generation, significant mortality occurred in adults of both sexes from the high-dose group (Table 1).
One animal in the P0 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 the test material and characteristic compound-induced lesions discussed subsequently were not noted.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Adult body weights were not affected in the low-dose groups (Table 1). 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 both sexes of the P0 generation were significantly reduced by Week 11, and this reduction persisted for the remainder of the test.

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 specified

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination of tissues from P0 adults revealed compound - related changes in the kidneys of rats from the high-dose group. Renal cortical necrosis, present in 60% of the males and in 15% of the females from this group, was not observed in any other groups.

Reproductive function / performance (P0)

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 (Table 2). However, there was neither a dose-related reduction nor a pattern within a generation that suggested the presence of a treatment-related effect.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

55 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

2

Dose descriptor:

LOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Respiratory rales also occurred in Fl rats, and the incidence and severity appeared to increase with dose.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The Fl 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 Fl generation tended to parallel that of the P0 generation except for the increase recorded for low-dose males.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Adult body weights were not affected in the low-dose groups (Table 3). The Fl generation showed a pattern that was roughly similar to the P0 generation, except that the only significantly depressed mean body weight occurred in high-dose males at 30 weeks.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In the Fl 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 specified

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no microscopic changes in these kidneys.

Details on results (F1)

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.

Effect levels (F1)

1

Dose descriptor:

NOEL

Generation:

F1

Effect level:

55 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

body weight and weight gain

other: litter size & pup survival

2

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: litter size & pup survival

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Results: F2 generation

General toxicity (F2)

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.

Details on results (F2)

Treatment with maleic anhydride had no effect on litter size or on pup survival at doses up to 55 mg/kg/day in the F2a and F2b litters (Table 3). Pup body weights in the experimental groups were significantly reduced at a few of the observation periods (Table 4). 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.

Effect levels (F2)

Dose descriptor:

NOEL

Generation:

F2

Effect level:

55 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: litter size & pup survival

Target system / organ toxicity (F2)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

TABLE 1. MORTALITY AND BODY WEIGHT OF ADULT RATS TREATED WITH MALEIC ANHYDRIDE DURING A MULTIGENERATION REPRODUCTION STUDY.

	Maleic anhydride (mg/kg/day)
	0	20	55	150
P0 generation
Males
Mortalitya	0(0)	10(10)	10 (0)	70b (60)b
Body weightc	143	142	143	143
	502	524	506	431b
	700	697	666	562b
Females
Mortalitya	0(0)	0(0)	5 (0)	65b (65)b
Body weightc	129	130	130	127
	289	281	280	259b
	368	345	337	317b
Fl generation
Males
Mortalitya	20(0)	40 (40)b	40 (0)	75b (58)b
Body weightd	113	107	103	85b
	495	500	445	431
	722	703	683	-
Females
Mortalitya	20(0)	30(5)	52b(10)	100b (14)
Body weightd	96	100	95	84
	265	272	265	247
	334	343	347	-

a Total dead/total treated X 100 (% mortality minus gavage-related deaths). Groups contained 10 to 12 males and

20 to 21 females.

b Significantly different from the appropriate control.

c Body weight (g/rat) at Weeks 0, 11, and 32 of the study.

d Body weights (g/rat) at Weeks 30,41, and 61 of the study.

TABLE 4. FERTILITY OF RATS TREATED WITH MALEIC ANHYDRIDE DURING A MULTIGENERATION REPRODUCTION STUDY.

	Maleic anhydride (mg/kg/day)
	0	20	55	150
Females
Fla	14/20(70)a	7/20(35)b	14/20(70)	7/20(35)b
Fib	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
Fla	8/10(80)c	5/10(50)	9/10(90)	4/10(40)b
Fib	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).

b Significantly different from control.

c Number fertile/number mated X 100 (% fertile).

TABLE 5. LITTER SIZE OF RATS TREATED WITH MALEIC ANHYDRIDE DURING A MULTIGENERATION REPRODUCTION STUDY.

Litter	Days after birth	Maleic anhydride (mg/kg/day)
		0	20	55	150
F1a	0	12.2a	11	11.6	13.1
	4	12.0(9.9)b	10.5(9.3)	11.2(9.3)	13.4(10.0)
	21	9.9	9.3	8.8	10
F1b	0	13.3	10.3	13.4	11.3
	4	13.0(9.8)	9.6 (9.0)	13.2(9.9)	10.8(9.7)
	21	9.8	8.9	9.8	9.3
F2a	0	13.4	12.2	12	-
	4	13.1(9.9)	11.6(10.0)	11.8(9.8)	-
	21	9.9	9.9	9.8	-
F2b	0	10.5	13.6	14	-
	4	10.4(8.2)	13.3(9.8)	13.8(10.0)	-
	21	8.2	9.7	9	-

a Mean live pups/litter on indicated day. The number of pregnant females that gave birth on Day 0 is presented in Table 4.

b Mean live pups/litter before litter reduction (mean live pups/litter after reduction to five pups/sex/litter when possible).

TABLE 6. BODY WEIGHT OF PUPS FROM RATS TREATED WITH MALEIC ANHYDRIDE DURING A MULTIGENERATION REPRODUCTION STUDY

Litter	Days after birth	Maleic anhydride (mg/kg/day)
		0	20	55	150
F1a	0	6.a	6.6	6.7	5.8*
	4	12(11)c	11(11)	12(11)	10 (9)
	21	58(56)	54(53)	58(55)	46* (44)*
F1b	0	6.4	7.1b	6.2	6.3
	4	11(10)	12(12)	11(10)	10 (10)
	21	53(50)	54 (54)	51(50)	47 (46)
F2a	0	6.6	6.4	6.7	-
	4	11(10)	10(10)	10(10)	-
	21	47(45)	47 (46)	46 (45)	-
F2b	0	6.8	6.4	6.1	-
	4	11(11)	10(9)	9(9)	-
	21	50(57)	45 (44)b	43 (44)b	-

a Mean weight (g) of both males and females.

b Significantly different from control.

c Mean weight (g) of males (mean weight (g) of females).

Applicant's summary and conclusion

Conclusions:

The reproductive NOEL for maleic anhydride was considered to be 55 mg/kg bw/day. A high degree of maternal toxicity was detected at the 150 mg/kg bw/day dose group which is considered to mask any effect on reproductive toxicity in this dose group.

Executive summary:

In the multigeneration study, 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. 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. The high-dose group was terminated during the second generation due to treatment-related mortality in adults. Renal cortical necrosis occurred in high-dose Fo males and females. Increased kidney weights were observed in low- and mid-dose adult Fl females. No treatment-related effects on reproduction were observed with maleic anhydride at doses up to 55 mg/kg/day over two generations.

The reproductive NOEL for maleic anhydride was considered to be 55 mg/kg bw/day.  A high degree of maternal toxicity was detected at the 150 mg/kg bw/day dose group which is considered to mask any effect on reproductive toxicity in this dose group.