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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethyl maleate
EC Number:
210-848-5
EC Name:
Dimethyl maleate
Cas Number:
624-48-6
Molecular formula:
C6H8O4
IUPAC Name:
dimethyl (Z)-but-2-enedioate
Test material form:
other: liquid
Details on test material:
Identification: DMM (dimethyl maleate, CAS 624-48-6)
Description: Clear liquid
Batch Number: LEDM8B7002
Purity/Concentration as Supplied: 99%
Purity/Concentration for Formulation: 100%
Correction for Purity: No
Stability of Test Item in Vehicle: Stable for up to 8 days.
Expiry Date: Sept-2013
Expiry Date (defined by Harlan): 30-Sept-2013
Storage Conditions: Room temperature (20 ±5 °C) away from direct sunlight. (Note: 18 - 36 °C according to email 22 Oct 2102)
Analytical Standard: The test item served as analytical standard/reference item.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
Age (at Start of Treatment): 11 weeks
Body Weight Range (at Acclimatization): Males: 315 to 358 g (mean 336 g); Females: 180 to 229 g (mean 198 g)
Identification: Cage card and individual animal number (ear tattoo). On day 1 post partum, pups were individually tattooed with Indian ink.
Randomization: Computer-generated random algorithm.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Husbandry
Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%).
The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Harlan Teklad 2018C rat / mouse maintenance diet was available ad libitum. The feed batch was analyzed for contaminants.
Water: Community tap-water from Itingen was available ad libitum in water bottles.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Daily Target Dose Level:
Group 1: 0 mg/kg/day
Group 2: 50 mg/kg/day
Group 3: 200 mg/kg/day
Group 4: 800 mg/kg/day (removed)
Group 5: 400 mg/kg/day

Rationale for Dose Level Selection: The dose levels were selected based on a previous
dose range finding toxicity study in Wistar rats.

Dose Volume: 10 ml/kg

Dose Concentrations:
Group 1: 0 mg/mL/day
Group 2: 5 mg/mL/day
Group 3: 20 mg/mL/day
Group 4: 80 mg/mL/day
Group 5: 40 mg/mL/day
Details on mating procedure:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation is observed. The females were removed and housed individually if:
- the daily vaginal smear is sperm positive, or
- a copulation plug is observed.
The day on which a positive mating is determined (copulation plug or sperm) was designated day 0 post coitum.
If a female does not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which has already mated successfully, were considered. If mating is not recorded during this additional pairing period of a maximum of 14 days, the female were sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.
All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum.
Day 0 were designated as the day on which a female has delivered all her pups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of Dose Formulations: For up to one week, based upon the results of stability analyses.
Storage of Dose Formulations: At room temperature (20 ± 5 °C) in glass beakers.

Analysis of Dose Formulations
The dose formulations were analyzed using a method provided by the Sponsor. The samples were delivered to the analytical laboratory. Transport of Dose Formulations to analytical laboratory: At ambient temperature (20°± 5 °C). Storage of Dose Formulations in analytical laboratory: Frozen (ca. -20 ± 5 °C).
Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start and concentration and homogeneity during week 6.

Duration of treatment / exposure:
Females
First Test Item Administration: Day 1 of pre-pairing. Pre-Pairing: 14 days
Treatment Ends: On day 3 post partum

Males
First Test Item Administration: Day 1 of pre-pairing. Pre-Pairing: 14 days
Treatment Ends: On day before sacrifice. After a minimum of 28 days treatment
Frequency of treatment:
Daily
Details on study schedule:
Study Sequence Females
Acclimatization: 5 days minimum
First Test Item Administration: Day 1 of pre-pairing
Pre-Pairing: 14 days
Pairing: 14 days maximum
Gestation: Approximately 21 days -
Treatment Ends: On day 3 post partum
Necropsy: On day 4 post partum

Study Sequence Males
Acclimatization: 5 days minimum
First Test Item Administration: Day 1 of pre-pairing
Pre-Pairing: 14 days
Pairing: 14 days maximum
Treatment Ends: On day before sacrifice
Necropsy: After a minimum of 28 days treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
0-50-200-400-800(removed) mg/kg/d
Basis:
nominal conc.
No. of animals per sex per dose:
Group 1: 12 males and 12 females
Group 2: 12 males and 12 females
Group 3: 12 males and 12 females
Group 4: 12 males and 12 females (removed *)
Group 5: 12 males and 12 females
Group 10: 2 males and 2 females
Total Number of Animals Used: 60 males and 60 females
Total Number of Animals Ordered: 62 males and 62 females

* Group 4 (800 mg/kg/d) animals were removed from the study for ethical reasons and Group 5 was added.
Control animals:
yes, concurrent no treatment
Positive control:
No.

Examinations

Parental animals: Observations and examinations:
- Viability / Mortality: Twice daily
- Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
- Food Consumption: Males: Weekly during pre-pairing and after pairing periods.
Females: Pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum. Food consumption was not recorded during the pairing period.
- Body Weights: Was recorded daily from treatment start to day of necropsy.
Litter observations:
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.
Postmortem examinations (parental animals):
Necropsy
Males were sacrificed after they were treated for approx. 43 days, or until no longer needed for mating.
If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital.
All P generation animals were exsanguinated.
All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurs to establish, if possible, the cause of death. For the parent animals, special attention was directed at the organs of the reproductive system.
Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
The numbers of implantation sites and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.

Organ Weights
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.
Postmortem examinations (offspring):
Dams and pups were sacrificed on day 4 post partum. All parent pups were examined macroscopically for any structural changes.
Statistics:
The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.
Reproductive indices:
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.
For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.
Offspring viability indices:
From the on-line recorded reproduction data, the following parameters were calculated: mean litter size, pup sex ratios and viability indices.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Higher rate of deaths and increased activity and salivation at 400 mg/kg/d
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced mean body weight of males and non-adverse transient reduced food consumption in males at 400 mg/kg/d
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced mean body weight of males and non-adverse transient reduced food consumption in males at 400 mg/kg/d
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Erosion, ulceration, and epithelial hyperplasia in the stomach of animals receiving 400 mg/kg/day
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Histological examination of spermatogenesis was perfeormed.
Reproductive performance:
no effects observed

Details on results (P0)

Mortality / Viability
One control male was found dead on day 1 of the pairing period. All other control males survived until scheduled necropsy at the end of the after pairing period. At 50 mg/kg/day, all males survived until the scheduled necropsy. One male treated with 200 mg/kg/day was found dead on day 13 of the pre-pairing period. All other males of this group survived until scheduled necropsy at the end of the after pairing period. At 400 mg/kg/day, one male was killed for ethical reasons on day 11 of pre-pairing. A second male was found dead on day 7 of the pairing period. All other males survived until scheduled necropsy at the end of the after pairing period. One female was sacrificed for ethical reasons on day 19 of gestation and two females found dead.

Clinical Signs
During the pre-pairing period of males, increased salivation (red or clear) noted with increasing frequency in the males treated with 200 or 400 mg/kg/day was considered to be a non-adverse test item-related finding. At the higher dose level, additional findings included increased activity and pica, which were also considered non-adverse test item-related findings, as was ruffled fur and which was noted with increasing frequency during the pre-pairing period. During the females’ pre-pairing period, no clinical signs of toxicological relevance were noted in control females or in females treated with 50 mg/kg/day or 200 mg/kg/day.
During the pairing period, increased salivation was noted in some males at 50 mg/kg/day and all males at 200 mg/kg/day. Although increased salivation was considered to be a test item-related effect, it is commonly observed when a test item with an unpleasant taste is administered by oral gavage, and therefore is not considered to be an adverse effect. No clinical signs were noted during the pairing period in females treated with 50 mg/kg/day or 200 mg/kg/day. At 400 mg/kg/day, findings in surviving males and females included increased activity and pica, as well as increased salivation, with ruffled fur with increasing frequency. One male and six females had transient general red staining of the lower mandible.
During the after pairing period, all males treated with 50 mg/kg/day had increased salivation at some point. All surviving males treated with 200 mg/kg/day had increased salivation at some point during the after pairing period. At 400 mg/kg/day, findings in all surviving males included salivation (one male had red salivation in addition), increased activity and pica during the after pairing period. Ruffled fur was noted in four males.
During the gestation period, increased salivation was noted in all surviving females at 50 mg/kg/day, 200 mg/kg/day and 400 mg/kg/day. General red staining of the lower mandible was noted at all dose levels with increasing incidence. All surviving females at 400 mg/kg/day showed increased activity, pica and intermittently ruffled fur during the gestation period.
During the lactation period, increased salivation was transiently increased in all surviving females at all dose levels. Ruffled fur was noted with increasing incidence in some females at all dose levels.

Food Consumption - Pre-Pairing and After Pairing Period
The mean food consumption of the males treated with 50 mg/kg/day or 200 mg/kg/day were similar. At 400 mg/kg/day, the mean daily food consumption of the test item treated males was initially lower than that of the control males (days 1 - 8 of the pre-pairing period). These minor differences were considered to be unrelated to the treatment with the test item.

Food Consumption - Pre-Pairing, Gestation and Lactation Periods
The mean food consumption of the test item-treated females generally compared favorably with those of the control females during the pre-pairing, gestation and lactation periods.

Body Weights - Pre-Pairing, Pairing and After Pairing Period
The mean absolute body weights of the males treated with 50 mg/kg/day or 200 mg/kg/day
generally compared favorably with those of the control males during the pre-pairing, pairing and after pairing periods. At 400 mg/kg/day, the males lost weight initially, but thereafter maintained or gained weight from day 8 onwards, but remained lower during days 5 - 14 of pre-pairing. During the pairing period, the body weights remained slightly lower than the control males. During the after pairing period, the mean absolute body weights remained lower than those of the control males. These differences were considered to be related to the treatment with the test item.

Body Weights - Pre-Pairing, Gestation and Lactation Periods
The mean absolute body weights of the females treated with 50 mg/kg/day or 200 mg/kg/day generally compared favorably with those of the control females during the pre-pairing, gestation and lactation periods.
At 400 mg/kg/day, the mean absolute body weights were higher than those of the control females from day 1 of pre-pairing onwards. This difference persisted through the gestation period. During the lactation period, the mean absolute body weights remained higher than those of the controls for three of the four measurements. However, this group of females was delivered after the other groups started the study, and were therefore not randomized at the same time as the other females, therefore these higher mean absolute weights were considered to be an artifact of the separate delivery; the mean body weight gain and differences in mean body weight gain were compared to the control values to ameliorate the differences in the absolute weights.

Mating Performance and Fertility
The mean precoital times of the test item-treated rats were similar in test item-treated and control groups. The mating rate, fertility index, conception rate and gestation index of the test itemtreated rats were unaffected. The duration of gestation was similar at all dose levels. The number of corpora lutea observed at all dose levels compared favorably with those of the controls.
The respective mean implantation rate and the mean post-implantation loss of the test itemtreated females were similar to that of the control females. The mean number of affected litters was similar in control and treated females. The mean litter sizes were similar in test item-treated and control females. Although the respective mean postnatal losses and the mean postnatal loss rate (as a percent of living pups) differed, the birth and viability indices of the females treated with 50 mg/kg/day and 200 mg/kg/day compared favorably with the controls. At 400 mg/kg/day, higher mean postnatal loss, number of affected litters and the total postnatal loss were noted at the end of the lactation period. These differences were considered to be related to the treatment with the test item. The viability index of the females treated with 400 mg/kg/day was lower when compared with the controls.

Organ Weights
In males, the mean absolute and relative organ weights were considered to be unaffected by the treatment with the test item.

Macroscopic Findings - Males
Males treated with 50 mg/kg/day and 200 mg/kg/day were without macroscopical findings. In two premature decedent males and two surviving males at 400 mg/kg/day, macroscopical findings were noted in the stomach and were considered to be largely related to local effects of the treatment with the test item.

Macroscopic Findings - Females
All control females and all females treated with 50 mg/kg/day and 200 mg/kg/day survived until scheduled necropsy. At 400 mg/kg/day, one female was sacrificed for ethical reasons on day 19 of gestation and had macroscopical changes to the forestomach and liver. Two females at 50 mg/kg/day, two females at 200 mg/kg/day, and two females at 400 mg/kg/day were sacrificed on day 25 of the gestation period and were without any macroscopical findings. All surviving females with litters were without findings of toxicological relevance.

Microscopic Findings
There were no treatment-related findings in reproductive organs.
In particular, qualitative examination of the stages of spermatogenesis in the testis did not reveal any treatment-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No treatment-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries.
There were treatment-related finding consistent with local irritation in the stomach of animals receiving the high dose of 400 mg/kg/day. These consisted of thickened/irregular stomach and crateriform retractions at necropsy, and erosion, ulceration, and epithelial hyperplasia at microscopic examination.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Findings at 400 mg/kg/d.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Higher mean postnatal loss, number of affected litters and the total postnatal loss at 400 mg/kg/day. Lower birth and viability indices in dams at 400 mg/kg/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower birth weights, reduced body weight development to day 4 post partum and lower mean pup weights on day 4 post partum at 400 mg/kg/day.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Reduced size of 5 pups at 400 mg/kg/d
Histopathological findings:
not examined

Details on results (F1)

Litter Data
At first litter check, one pup at 200 mg/kg/day was found dead from litter neglect and was cyanotic. Four pups of one litter at 400 mg/kg/day were found partly cannibalized.
During observations during days 1 - 4, two pups of litter 61 (50 mg/kg/day) were found dead and had cleft palate. A second pup of litter no 82 (200 mg/kg/day) was missing its tail on day 4 of lactation. On days 2 - 4 of lactation, scabbing was noted on the left flank of pup no 9 of litter 122. On day 4, pups 1 - 10 of litter 124 (400 mg/kg/day) were found to be neglected.
The sex ratios at first litter check were within the range of typical variation. The mean body weights of the pups from litters of dams treated with 50 mg/kg/day and 200 mg/kg/day were similar to those of the control litters, but the birth weights of the pups at 400 mg/kg/day were clearly lower and the body weight development to day 4 post partum was markedly lower than those of the controls. The mean pup weights were lower on day 4 post partum when compared with pups of the control group; these differences were considered to be related to the treatment with the test item.
There were no macroscopical findings in the control pups or in those of dams treated with 50 mg/kg/day and 200 mg/kg/day. At 400 mg/kg/day, five pups were found to be reduced in size and one was found to have skin sores.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Findings at 400 mg/kg/d.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for the subacute toxicity part of the study, as well as for the parental and the offspring effects is 200 mg/kg/d.
Executive summary:

Investigations according to the method OECD421 of the subacute toxicological effects resulting from repeated oral-gavage administration of dimethyl maleate, CAS 624-48-6 to parental rats were performed. Dimethyl maleate was administered at dosages of 50, 200, and 400 mg/kg body weight/day, and controls received the vehicle water only. Dimethyl maleate was administered to male rats for 44 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Marked signs of systemic toxicity was noted in rats treated with 800 mg/k/day, this group was therefore not followed.

Although there were no test item-related deaths, there was a non-adverse transient reduction of mean daily food consumption in males treated with 400 mg/kg/day. There were no effects on the mean absolute or relative organ weights of parent animals, no effects on mating performance and fertility (including mean precoital times, mating rate, fertility index, conception rate and gestation index, duration of gestation or number of corpora lutea), mean implantation rate and the mean post-implantation loss, mean number of affected litters and mean litter sizes, or sex ratios. There were no test item-related macroscopical findings in the offspring at any dose level. There were no treatment-related findings in reproductive organs.

 

Test item-related findings included increased salivation at all dose levels (which was considered to be not adverse), and increased activity at 400 mg/kg/day, reduced mean body weight development in males treated with 400 mg/kg/day, higher mean postnatal loss, number of affected litters and the total postnatal loss at 400 mg/kg/day, lower birth and viability indices in dams at 400 mg/kg/day, lower birth weights, reduced body weight development to day 4 post partum and lower mean pup weights on day 4 post partum at 400 mg/kg/day. There were treatment-related finding consistent with local irritation in the stomach of animals receiving the high dose of 400 mg/kg/day. These consisted of thickened/irregular stomach and crateriform retractions at necropsy, and erosion, ulceration, and epithelial hyperplasia at microscopic examination.

The NOEL (No Observed Effect Level) for general toxicity in males and females was considered to be below 50 mg/kg/day, but the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females was considered to be 200 mg/kg/day.

The NOEL (No Observed Effect Level) and the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was considered to be 200 mg/kg/day.

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