Dioctyl maleate, branched

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The multi-generation study available on the read-across substance contains multiple methodological dificiencies and as such cannot be used for classification and labelling of the registered substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Reference 2

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.

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

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%

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

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.

Dose / conc.:

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

Dose / conc.:

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

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.

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.

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: 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.

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

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

Critical effects observed:

not specified

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.

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:

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

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

Critical effects observed:

not specified

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.

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.

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

Critical effects observed:

not specified

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

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).

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.

Effect on fertility: via oral route

Endpoint conclusion:

no study available (further information necessary)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available (further information necessary)

Effect on fertility: via dermal route

Endpoint conclusion:

no study available (further information necessary)

Effects on developmental toxicity

Description of key information

The key study was conducted on a read-across substance with substantially similar breakdwon products. The study was conducted before adoption of the relevant OECD testing guideline and OECD GLP standards. The method of the study is equivalent to the relevant OECD testing guideline.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Remarks:

Study published before adoption of OECD guideline.

GLP compliance:

no

Remarks:

Study published before adoption of GLP standards.

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%

Species:

rat

Strain:

other: CD

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

Route of administration:

oral: gavage

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. A 1% (w:v) concentration was used to administer all doses. Rats (25 mated females/group) were treated orally with 30, 90, or 140 mg/kg/day of maleic anhydride. Mated females in the control group were treated in a similar manner with 14 ml/kg of corn oil.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

One male and one female were housed together for mating. The day of mating was determined by daily inspection for a copulatory plug or a sperm-positive vaginal smear. This day was designated Day O of gestation.

Duration of treatment / exposure:

Day 6 through Day 15 of gestation.

Frequency of treatment:

Daily

Duration of test:

To day 20 of gestation.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25 mated females

Maternal examinations:

Body weights were recorded at intervals during gestation.

Fetal examinations:

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 hydroxide, stained with Alizarin Red S (Dawson, 1926), and examined for skeletal abnormalities.

Clinical signs:

no effects observed

Description (incidence and severity):

The general appearance and behavior of rats were not altered by treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

While one adult died in each of the experimental groups, the overall survival in these groups was 96% (Table 1).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dams in the experimental groups either failed to gain weight or lost weight between Days 6 and 9 of gestation (Table 1). However, this effect was reversible, and there were no statistically significant effects on body weight at any of the times examined.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no evidence of postimplantation loss (Table 1).

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

clinical signs

mortality

pre and post implantation loss

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

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 concurrent control and all treated groups were slightly greater than the values for historical controls.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Dams from all test groups produced normal-sized litters (Table 1).

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The fetal variations were comparable both in type and frequency in the control and treated groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The fetal variations were comparable both in type and frequency in the control and treated groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

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 (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: tail

external: pelvic region

skeletal: skull

skeletal: sternum

visceral/soft tissue: musculoskeletal system

Developmental effects observed:

no

TABLE 1. MATERNAL AND FETAL DATA FROM RATS TREATED WITH MALEIC ANHYDRIDE DURING GESTATION

	Maleic anhydride (mg/kg/day)
	0	30	90	140
Number treated	25	25	25	25
Pregnant	23	24	20	21
Alive	23	23	19	21
Nonpregnant	2	1	5	4
Alive	2	1	5	3
Body weight (g)
Day 0	242	244	242	243
Day 6	261	264	266	264
Day 9	272	264	264	258
Day 12	293	286	283	282
Day 15	315	302	303	298
Day 20	397	377	385	378
Implants/dam	13.3(14)a	13.4(14)	13.2(13)	13.5(14)
Viable fetuses/dam	12.6(13)	12.7(13)	12.5(13)	12.7(13)
Resorptions/dam	0.8 (0)	0.7(0)	0.7(1)	0.8 (0)
Fetal weight (g)	4.0(3.8)	3.7(3.8)b	3.8(3.8)	3.7 (3.6)b

a Mean (median).

b Mean significantly different from control. The historical control value for fetal weight was 3.6 g for 1774 fetuses

TABLE 2. OBSERVATIONS OF FETUSES FROM RATS TREATED WITH MALEIC ANHYDRIDE DURING GESTATION

	Maleic anhydride (mg/kg/day)
	0	30	90	140
Number examined
Litters	23	23	19	23
Fetuses
External	289	292	237	267
Skeletal	189	190	155	174
Soft tissue	100	102	82	93
Malformations
Short tail		1 (1)a
Omphalocele				1 (1)
Spherical enlargement on ribs	1 (1)
Fused sternebrae		1 (1)
Bent ribs				1 (1)
Multiple anomalies				1 (1)b
Total with above malformations	1 (1)	2 (2)	0 (0)	3 (3)
Variations
Rudimentary 14th rib(s)	51 (17)	62 (20)	46 (16)	37 (13)
Full 14th rib(s)	1 (1)	2 (2)	3 (3)	1 (1)
27 presacral vertebrae		5 (4)	2 (1)	2 (2)
Sternebrae 5 and/or 6 unossified	12 (9)	16 (7)	12 (6)	20 (12)
7th cervical rib	3 (2)	1 (1)	1 (1)
Misaligned centra				1 (1)
Reduced ossification of skull		1 (1)		1 (1)
Hyoid unossified		5 (3)
Pubis unossified		1 (1)
Renal papillar not developed	2 (2)			1 (1)

a Number of fetuses (number of litters).

b Includes malformed humerus, stemoschisis, and ossification defects of the pubis, cervical arches, and skull

Conclusions:

Under the conditions of this study, it is concluded that maleic anhydride is not teratogenic.

Executive summary:

In the teratology study, pregnant rats (19-23/group) 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 skeletaldefects. A reduced weight gain or weight loss was observed in all maleic anhydride-treated groups between Days 6 and 9; however, mean weights of all groups were within 5% of control on Days 15 and 20. No treatment-related effects on fetal development were observed.

Reference 2

Endpoint:

developmental toxicity

Type of 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

Justification for type of information:

Full read-across information is appended.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source substance and target substances behave in substantially similar ways in water, and are considered to be functionally similar when inside the body. The target substance has a higher molecular weight and lower dermal absorption coefficient, and is therefore considered to be less likely to enter the body through the skin or via oral absorption. The potential for acute dermal toxicity, repeated dose toxicity and toxicity to reproduction is therefore lower in the target substance than the source substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Both substances are esters, with the carbonyl (C=O) part of each ester separated by 2 carbons. In addition, the two carbons are unsaturated as they share a double bond. In both cases this double bond is present in the cis geometry. The principle difference between the two substances is that the maleic anhydride is a ring structure, with the esters sharing the hydroxy group between them; whereas in the target substance there are two distinct ester components with branching chains (a methyl (odd) and a hexyl (even)).
Both substances can undergo hydrolysis to produce the same carboxylic acid as shown in the appended .pdf (Table 4). However, Maleic anhydride undergoes this process more readily as it is hygroscopic. The carboxylic acid formed is maleic acid, which can undergo hydration upon further reaction with water to produce malic acid.
The principle difference between how the source and target substances undergo hydrolysis is that one mole of maleic anhydride only produces one mole of maleic acid and no other organic species, whereas one mole of the target substance bis(1-methylheptyl) maleate produces one mole of maleic acid and two moles of 2-octanol. The 2-octanol is a branched alcohol, with an odd numbered branch (due to the methyl chain) and an even numbered branch (due to the hexyl chain).

3. ANALOGUE APPROACH JUSTIFICATION
Due to the similarities of the source and target substance with regards to chemical structure, physico-chemical properties, and Lipinski’s rule of 5, the target substance is expected to behave in a substantially similar manner in vivo.
The target substance is therefore predicted to also have a reproductive NOEL of >55 mg/kg/day after repeated oral dosing in the rat. The target substance is also predicted to show no treatment-related effects on fetal development at up to 140 mg/kg/day after repeated oral dosing in the rat. By extension, the target substance is considered not to fulfil the criteria for toxicity to reproduction under the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

4. DATA MATRIX
See appended read-across justification.

Reason / purpose for cross-reference:

read-across source

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

clinical signs

mortality

pre and post implantation loss

Abnormalities:

no effects observed

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: tail

external: pelvic region

skeletal: skull

skeletal: sternum

visceral/soft tissue: musculoskeletal system

Developmental effects observed:

no

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:

2

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based upon the information presented in the key study, the registered substance does not meet the criteria for classification as toxic to reproduction in accordance with the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).