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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A screening study according to OECD 421 with the registered substance, 4-MHHPA, showed no adverse effects on fertility. The NOAEL was determined to be 450 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Well described GLP compliant study conducted to recognised international test guidelines.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Key study


 


OECD 443 (2023)


An Extended One Generation Reproduction Toxicity Study according to OECD 443 is on-going. Detailed information will be provided after finalization. 


 


OECD 421 (2010)


The effects hexahydro-4 methylphthalic anhydride (4-MHHPA) on fertility, pregnancy and early lactation of the offspring have been investigated in a reproduction/developmental toxicity screening study conducted according to OECD test methods. Groups of rats were dosed by oral gavage at levels of 50, 150 and 450 mg/kg/day. Males were treated for 2 weeks prior to pairing and during pairing of all females until the day before necropsy, for a total of 6 weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods and for 4 days post partum. No significant changes and no treatment-related effects were detected in males and females during the in vivo phase. Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls. Gestation length, litter data, sex ratios and implantation losses (pre and post) were unaffected by treatment. Findings at macroscopic and microscopic examinations, in both males and females, did not reveal differences between groups that could be related to treatment.


On the basis of these findings, 450 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL). This study is acceptable and satisfies the guideline requirement for a reproduction and developmental toxicity screening study in rats (OECD 421).


 


Supporting study


 


OECD TG 443 DRF (similar to OECD 421; 2022)


This study was conducted to determine the initial information on the toxic characteristics, systemic toxicity, and reproduction/or developmental toxicity of the test item and to select dose levels for a future extended one-generation reproductive toxicity study (EOGRTS) when administered orally in male and female Wistar rats, through gavage, during pre-mating, mating, gestation, and lactation periods.


In the parental generation (P) 8 rats/sex/group and in the filial generation (F1) 8 rats/sex/group were treated from postnatal day (PND) 21 to 35. 


Based on the existing toxicity data the following dose levels were initially selected for the study: 200 (G2), 400 (G3), and 800 (G4) mg/kg bw/day. As treatment-related mortalities were observed in both sexes (8/16) at 800 mg/kg bw/day (G4) by treatment day 5, the dose was reduced to 600 mg/kg bw/day from treatment day 5 for the surviving rats. However, the surviving rats (treated at 600 mg/kg bw/day) were also either found dead or moribund sacrificed by treatment day 6.
As all rats were found dead in the G4 treatment group, an additional group (G5) was included in the study which was treated at 600 mg/kg bw/day. As again, treatment-related mortalities were observed in both sexes (11/16) at 600 mg/kg bw/day by treatment day 6, the dose was further reduced to 500 mg/kg bw/day from treatment day 7 for the surviving rats. However, the surviving rats (treated at 500 mg/kg bw/day) were also either found dead or moribund sacrificed by treatment day 8 except three female rats which were humanely sacrificed on treatment day 12, as no male rats were available for the mating purpose.


Therefore, the control group (G1), 200 (G2) and 400 mg/kg bw/day (G3) were used for further study performance. Males were treated 46 days (4 weeks prior to mating, during the mating (two weeks) until the day before scheduled sacrifice), females were treated 4 weeks prior to mating, the variable time to conception, the duration of pregnancy and 21 days after delivery. Selected F1 were treated from PND 21 to 34.


Examination parameters were as follows:


Mortality & Morbidity in P and F1, Clinical Signs in P and F1, Body Weight, Food Consumption, Oestrus Cycle, Pup Observation, Blood Collection, Lacteal Transfer, Gross Necropsy, Organ Weights. 


Results:


No treatment-related effects were observed for any evaluated parameter in G2. 


In G3, the following treatment-related effects were observed:



  • Two mortalities (1/8 male and 1/8 female rat).

  • Intermittent salivation in male (7/8) and female (8/8) rats.

  • Rales in male (4/8) and female (5/8) rats for approximately 1-3 weeks of treatment, the recovery was noted after the 3 weeks treatment period.

  • Weakness in male (1/8) and female (3/8) rats.

  • Statistically, a significant decrease in mean body weight gain was observed during premating days 1-8 in male rats. Similarly, a decrease in mean body weight gain without statistical significance was also observed in female rats during pre-mating days 1-8. This decrease in the mean body weight gain of male and female rats was observed during the first week of treatment (premating days 1-8) after that rats were adapted to the treatment and recovery in mean body weight gain was noted. This effect was considered as related to the test item treatment.

  • Statistically, a significant decrease in overall mean food consumption of male rats was observed compared to that of the control group. Individually and statistically, a significant decrease in mean food consumption was also observed during premating days 8-15 and 15-22. These decreases in the mean food consumption of male rats were considered an effect of the test item treatment.


At 600/500 mg/kg bw/day test item (G5) the following treatment-related effects were observed:



  • Twelve mortalities (7/8 male and 5/8 female rats)

  • One male rat was sacrificed under moribund conditions.

  • Intermittent salivation in male (7/8) and female (3/8) rats.

  • Rales in male (2/8) and female (1/8) rats.

  • Weakness in male (6/8) and female (5/8) rats.

  • Piloerection in male (5/8) and female (3/8) rats.

  • On gross (macroscopic) examination, red discolouration was observed in the stomach of 7 males and 5 females.


At 800/600 mg/kg bw/day (G4) the following treatment-related effects were observed:



  • Eleven mortalities (4/8 male and 7/8 female rats)

  • Four male and one female rat were sacrificed under moribund conditions.

  • Intermittent salivation in male (8/8) and female (6/8) rats.

  • Rales in male (6/8) and female (6/8) rats.

  • Weakness in male (5/8) and female (3/8) rats.

  • Piloerection in male (5/8) and female (2/8) rats.

  • On gross (macroscopic) examination, red discolouration was observed in the stomach of all 8 rats of either sex.


In G2 and G3 pups treated from PND 21 - 34,


No treatment-related effect was seen for any evaluated parameter, which includes:



  • Clinical signs and mortality

  • Mean body weight, body weight gain, and food consumption

  • Gross (macroscopic) examination of pups 


Lacteal transfer:


Results of the milk analysis revealed that MHHPA most likely in its hydrolysed form as the dicarboxylic acid "4-methylhexahydrophthalic acid" was transferred via milk and that exposure to the pups increased dose-dependently.


 


Based on the results of this study, under the condition and procedures followed dose levels of 800/600, 600/500 and 400 mg/kg bw/day produced severe toxicity in terms of treatment-related mortality, morbidity, and adverse clinical signs (rales, weakness, piloerection).
Considering these findings of MHHPA, the following dose levels are suggested for future extended one-generation reproductive toxicity study:
Low Dose: 90 (mg/kg bw/day)
Mid Dose: 180 (mg/kg bw/day)
High Dose – I: 270 (mg/kg bw/day)
High Dose – II: 360 (mg/kg bw/day)

Effects on developmental toxicity

Description of key information

Based on a prenatal developmental toxicity study in rats, conducted in accordance with OECD TG 414, the NOAEL is considered to be 460 mg/kg/day for development of offspring.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
460 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Well described GLP compliant study conducted to recognised international test guidelines.
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

Key study:


 


OECD 414 (2021)


Hexahydro-4-methylphthalic anhydride (4-MHHPA) was examined for its possible prenatal developmental toxicity in a study according to OECD 414. Groups of 28 to 30 sperm-positive female Han: of Wistar origin rats were treated with 4-MHHPA by oral administration daily at three dose levels of 460, 615 and 800 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 28 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 4 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item in sunflower oil was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 °C) at the concentrations of 1 and 280 mg/g. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 91 and 105 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. External fetal sex was determined by gross pathological examination and compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue alterations). The anogenital distance was measured. In addition, indication of incomplete testicular descent / cryptorchidism was noted in male fetuses. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.


In total, on gestation day 20 there were 23, 21, 19 and 20 evaluated litters in the control, 460, 615 and 800 mg/kg bw/day groups, respectively. One female died in the course of the study in the 800 mg/kg bw/day group due to an effect of the test item after clinical signs such as hunched back, piloerection and reduced activity and digging in the bedding in the days before. Necropsy revealed macroscopic changes in the lungs, liver and ileum. Histopathology supported these findings and additionally there were lesions in the heart observed. Similar test item related clinical signs were observed in the 800 and 615 mg/kg bw/day group. Reduced food consumption and lower body weight gain (including corrected body weight gain in the 800 mg/kg bw/day dose group) was observed in the 800 and 615 mg/kg bw/day group which was attributed to an effect of the test item. Slight reductions in the food consumption of the 460 mg/kg bw/day group animals were not considered as adverse. There were no significant differences in the body weight (including corrected body weight) of the dams. Lower values compared to control were measured in all dose groups (in similar manner) for the free thyroid hormone level FT4. The FT3 values were also statistically significantly lower in the 615 and 460 mg/kg bw/day groups. In the absence of a dose-response relationship and since the values were within the value of the historical control data these findings are considered not test-item related. The TSH hormone levels were similar in all groups. There were no test item related differences in thyroid weight among the dosing groups. The treatment did not result in histological changes of the thyroid gland in any of the dose groups. Slight increase of post-implantation loss (early and late resorptions) including the occurrence of total post-implantation loss in one female both in the 800 and 615 mg/kg bw/day group were likely attributed to an effect of the test item, possibly due to maternal toxicity. The fetal weight was slightly lower and the incidence of growth retarded fetuses was higher in the 800 and 615 mg/kg bw/day groups (at a similar level), however statistical analysis revealed no significance. Hence, a test item effect was not proved. There were no significant differences in the ano-genital distance and placental weight parameters. At fetal examinations, there were no significant differences in the incidence of malformations at any dose level. There were no malformed fetuses found on external examination. In one litter, the placentas were observed as larger and or with wider margin in the 800 mg/kg bw/day group. Considering the lack of dose response or the low incidences, the visceral variations observed were not attributed to treatment. Two single cases of brain malformations such as an external hydrocephaly and a hole in the thalamus region in the dose groups of 800 and one 615 mg/kg bw/day group were considered incidental and toxicologically not relevant. Some increases in delayed ossifications might be related to maternal toxicity in the 800 and 615 mg/kg bw/day groups. Slightly bent scapula occurred in the test item treated groups with low incidences (1 to 3 fetuses per dose group) and two fetuses of one litter had bent scapula (one with slightly thicker humerus) in the 800 mg/kg bw/day group (latter in the litter with placenta alterations). Slightly bent or bent scapula as well as malformed humerus may occur in control fetuses according to the background data (laboratory's HCD: slight wavy ribs: 16.1%), hence a test item response was not proved.


In conclusion the oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with 4-MHHPA at the dose level of 800 mg/kg bw/day caused test-item related death of one animal. Clinical signs of animals in the 800 and 615 mg/kg bw/day groups were attributed to the treatment. The treatment with the test item caused reduced food consumption and body weight gain in the 615 and 800 mg/kg bw/day groups. Besides a significantly decreased corrected body weight gain in the 800 mg/kg bw/day group no significant differences in the body weight/corrected body weight values were detected. Thyroid-related parameters (FT3, FT4 TSH, weight, histopathology) were not affected by treatment. The slightly but statistically significantly increased postimplantation loss in the 615 and 800 mg/kg bw/day group is considered to be treatment-related. Although the incidence of body weight retarded fetuses was slightly increased in the mid and high dose group, there were no statistically significant differences in the mean body weight of the fetuses. No statistically significant differences in the incidence of malformations over all dosing groups were observed. Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as to be 460 mg/kg bw/day for maternal toxicity and developmental toxicity including teratogenicity.


 


 


Supporting studies:


 


OECD 414 (2018)


Hexahydro-4-methylphthalic anhydride (4-MHHPA) was examined for its possible prenatal developmental toxicity in a GLP compliant study according to OECD 414. Groups of 26 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 80, 240 and 460 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 4 mL/kg bw. Concentrations of the test item in the dosing formulations varied in the acceptable range between 99 and 109 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. The body weights and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded. In total, on gestation day 20 there were 20, 19, 21 and 17 evaluated litters in the control, 80, 240 and 460 mg/kg bw/day group respectively. None of the females died before scheduled necropsy and there were no test item related clinical signs recorded in all dose groups. No treatment related necropsy findings were observed. There were no treatment related changes in food consumption or body weights indicated. The number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. There were no test item related adverse effects on the fetal weight, external and visceral development of fetuses. There were no test item related malformations found. Based on these observations the No Observed Adverse Effect Level (NOAEL) for both maternal and developmental toxicity (including teratogenicity) was 460 mg/kg bw/day.


 


DRF (2021)


Hexahydro-4-methylphthalic anhydride (4-MHHPA) was examined for its possible prenatal developmental toxicity in a dose range finding study. Groups of eleven to twelve spermium-positive female Han: WIST rats were treated with the test item by oral administration daily at three dose levels of 500, 700 and 900 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of eleven spermium positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 4 mL/kg bw. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when spermium was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day (g.d.) 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were examined externally.


On gestation day 20, a total of 10, 9, 11 and 6 litters in the control, 500, 700 and 900 mg/kg bw/day groups, respectively, were evaluated. The dose level of 900 mg/kg bw/day caused death of three dams. One dam died after five, one after six and one after fourteen administrations. Reduced activity and piloerection were recorded for the animals that died and in addition in two females that survived (one with total post-implantation loss and one non-pregnant) in the 900 mg/kg bw/day group. Laying down and dyspnoe was recorded for one female which died. Digging in the bedding after treatment and salivation was observed in all test item treated groups. All of the three animals which died before scheduled necropsy had dark red liver. Bloody or yellow or yellowish-brownish content in the intestines or yellow dilute or whitish content in the stomach was recorded for the animals that died and for the surviving female with total post-implantation loss in the 900 mg/kg bw/day group. Sporadically gas filled intestine segments were observed in the animals in the 900 and 500 mg/kg bw/day group. In one high dose animal which died reddish mottled lungs were found. Slight to moderately lower mean food consumption of the dams was indicated, most notable between g.d. 5 and 11, in the 900 mg/kg bw/day group. In the 700 mg/kg bw/day dose group the average food consumption was slightly lower between g.d. 5 and 11. This was not unambiguously attributed to the test item. Slightly lower body weight was observed in the 900 and 700 mg/kg bw/day group, in the latter not proved to be due to the test item. Differences were more notable in regard to lower body weight gain values in the 900 mg/kg bw/day dose group and also observed in the 700 mg/kg bw/day dose group. In the 700 mg/kg bw/day dose group this effect was less pronounced. The slightly lower body weight gain in the 500 mg/kg bw/day between g.d. 5 and 8 was not proved to be due to the test item. The corrected body weight gain was lower in the 700 and 900 mg/kg bw/day group with a dose dependent response. There was no dose response indicated in the mean percent of early embryonic death. The percentage of late embryonic death increased slightly and the average fetal death increased due to one female with total post-implantation loss in the 900 mg/kg bw/day group. There was no test item effect indicated in the number of implantations or percent pre-implantation loss. The number of viable fetuses was slightly to moderately lower in the 700 and 900 mg/kg bw/day group, not proved to be due to the test item at 700 mg/kg bw/day. Slightly lower fetal weight in the 900 mg/kg bw/day group and in case of female fetuses in the 700 mg/kg bw/day group was not considered as significant. There were no malformations or placental alterations found. There was no test item effect indicated in incidence of variations i.e. growth retardations.


In the present dose range finding study, oral treatment of pregnant Han: WIST rats with the dose level of 900 mg/kg bw/day of the test item caused death of three dams and clinical signs such as reduced activity, piloerection, laying, dyspnoe and salivation of the animals. The test item caused moderate reduction in the food consumption and lower body weight parameters at 900 mg/kg bw/day (including a minimal weight loss between g.d. 5 and 8 and a moderately lower corrected body weight gain). Less significantly lower food consumption and body weight parameters including corrected body weight gain at 700 mg/kg bw/day were not unambiguously attributed to an effect of the test item. The increase of fetal death in the 900 mg/kg bw/day dose group due to one female with total post-implantation loss and moderately lower mean number of viable fetuses was considered treatment related, most likely secondary to the severe maternal toxicity demonstrated by the strong clinical symptoms. The mean number of implantations, percentage preimplantation loss, early and late resorptions were not proved to be affected by the test item. Slightly lower body weight of fetuses was not proved to be due to the treatment with the test item. The test item caused neither malformations, nor increase of fetal variations or placental abnormalities. Based on the observations the dose range finding study determined the doses of 460, 615 and 800 mg/kg bw/d for the main study (OECD 414 (2021)). The No Observed Adverse Effect Level (NOAEL) was considered to be 500 mg/kg bw/d for maternal toxicity and developmental toxicity.


 


DRF (2018)


Hexahydro-4-methylphthalic anhydride (4-MHHPA) was examined for its possible prenatal developmental toxicity in a dose range finding study for a full prenatal developmental toxicity study (OECD 414). Groups of seven spermium-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000/460 mg/kg bw/day (the dose level of the high dose was reduced after mortality was observed) respectively from day 5 up to and including day 19 post coitum. A control group of seven spermium positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 4 mL/kg bw. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when spermium was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally.


The dose level of 1000 mg/kg bw/day of the test item caused clinical signs and death of two dams after treatment from gestation day 5 to 7. The test item caused death in a further dam after two treatments with 1000 mg/kg bw/day and one treatment with the reduced 460 mg/kg bw/day dose. The death of one dam in 300 mg/kg bw/day dose group was caused by misgavage and therefore is not test item related. During treatment from gestation day 5 to 19 with the dose level of 1000/460 mg/kg bw/day the test item caused clinical signs in one dam and in one female with total implantation loss even after reduction of the dose level. Similar clinical signs and necropsy findings were observed in one animal in the 300 mg/kg bw/day dose group. A test item related effect at 300 mg/kg bw/day was not considered due to the low incidence. There was no reduction in the food consumption and body weight parameters attributed to the test item considering the lack of dose response. The increase of early embryonic death in the 1000/460 mg/kg bw/day dose group was caused by one female with total post-implantation loss. The effect was considered treatment related, most likely secondary to the severe maternal toxicity demonstrated by the strong clinical symptoms. The mean number of implantations, viable fetuses per litters, percentage pre-implantation loss, late resorptions and fetal death were not affected by the test item. The treatment of the maternal animals did not result in reduced body weight of fetuses or increase of variations including body weight retardation. The test item caused no malformations in the fetuses.


Based on the maternal toxicity and effects secondary to maternal toxicity the dose range finding study determined the doses of 80, 240 and 460 mg/kg bw/d for the main study (OECD 414 (2018)). The NOAEL of the DRF study was determined to be 300 mg/kg bw/d based on maternal toxicity.


 


Conclusion:


As an overall conclusion adverse effects caused by the test item in the dams and the effects regarding the dams and offspring, possibly due to the maternal toxicity, have been identified and the NOAEL for maternal and developmental toxicity including teratogenicity was determined to be 460 mg/kg bw/day for hexahydro-4-methylphthalic anhydride (4-MHHPA).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008


The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008, as amended for the eighteenth time in Regulation (EU) 2022/692.


 


The results of reproductive and developmental toxicity testing show that 4-MHHPA classification and labelled according to Regulation (EC) No 1272/2008 (CLP) is not required.

Additional information