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Effects on fertility

Description of key information

One-generation reproduction study in rats: no effects on fertility (CEFIC, 1988, comparable to OECD guideline 415)

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Specific Pathogen Free (SPF) colony at the Alderley Park Breeding Unit, ICI
- Age at study initiation: 28 days
- Weight at study initiation: (P) Males: 72.5 g; Females: 71.1 g
- Housing: 2 females or 1 male per cage
- Diet (e.g. ad libitum): CTI diet supplied by Special Diets Servies Limited
- Water (e.g. ad libitum): filtered tap water
- Acclimatisation period: 6-7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 21
- Humidity (%): 45-60
- Air changes (per hr): 15 - 25
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 3-4 August 1987 To: 12 January 1988
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food):
Dose level 300 ppm: 9.07g/30 kg
Dose level 1800 ppm: 54.44g/30 kg
Dose level 12000 ppm: 362.90g/30 kg
Details on mating procedure:
- M/F ratio per cage: 1 male and 2 female per cage
- Length of cohabitation: 10 days
- Proof of pregnancy: vaginal smear were examined daily
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): separately
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Mean concentrations within 2% of target concentration for all groups. DEHA was not detected in any control diet (detection limit 10ppm). Chemical stability of DEHA in diet was determined on three batches of diet at nominally 300 ppm and 12000 ppm. Satisfactory chemical stability was established.
Homogeneity of DEHA in diet mixtures was satisfactorily demonstraded on the first diet batch at nominally 300 and 12000 ppm DEHA.
Duration of treatment / exposure:
Males: 10 weeks premating + mating period
Females: 10 weeks premating, mating, gestation (app. 22 days), lactation (22 days)
Frequency of treatment:
The rats in each generation were fed experimental diets continuously until termination.
Remarks:
Doses / Concentrations:
0, 300, 1800, 12000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 28, 170, 1080 mg/kg
Basis:
nominal in diet
No. of animals per sex per dose:
30 females and 15 males per group in total 4 groups.
Control animals:
yes, plain diet
Details on study design:
The dose leveis for this study were based on data from the literature (NTP, 1982) and included an anticipated no effect level and a level at which toxic effects of DEHA were expected at some stage during the course of the study.
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes, at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes, once weekly

BODY WEIGHT: Yes, of all rats were recored at weekly intervals throughout the premating period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, each cage of rats was recorded throughout the premating periods and calculated on a weekly basis. The food utilisation value per cage was calculated as the weight gained by the animals in the cage per 100g of food eaten.
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
no data
Litter observations:
STANDARDISATION OF LITTERS
A count of all live and dead pups was made within 24 hrs (day 1), at days 5, 11, 22, 29 and 36 post partum. The sexes of the pups were also recorded at these times.
Postmortem examinations (parental animals):
SACRIFICE
All animals at scheduled kills and those killed during the study were anaesthetised by inhalation of halothane BP vapour and killed by exsanguination. All surviving males were killed after completion of mating. All females were killed after weaning thier litters.

Histological examination: Cervix, Epididymis, Liver, Mammary gland, Ovary, Prostate, Seminal vesicle, Testis, Uterus, Abnormal tissues.
Postmortem examinations (offspring):
All pups were killed as soon as possible after Day 36 post partum.
Statistics:
Mean bodyweight gain, food consumption and food utilisation during the premating period, female bodyweight gain during pregnancy, parental liver weights and pup (litter) bodyweight gain until Day 36 post partum.
Reproductive indices:
Mean lenght of gestation, mean pre-coital interval
Offspring viability indices:
Mean live born index, mean survival index, mean litter size, total litter weight and whole litter losses.
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment related abnormalities

BODY WEIGHT
Bodyweight gain was marginally reduced for high dose females. The difference was statistically significant during pregnancy weeks 2 (-15%) and 3 (-10%). Body weight was also significantly reduced (-6%) at the end of pregnancy (intermediate data not given). Body weight for parental females was not included in the study report for the lactation period. There was no effect on body weight gain in any other treatment group.

TEST SUBSTANCE INTAKE
There was a slight increase in food consumption in males dosed with 12000ppm DEHA from 6-10 weeks of the study, the effect being statistically significant at weeks 6-9. Food utilisation was slifghtly less efficients overall for males receiving 12000ppm DEHA.

REPRODUCTIVE PERFORMANCE
There was no effect on male or female fertility, gestation length, and pre-coital interval in any dose group. Litter size was slightly and not significantly reduced in the high dose group, but because of the minimal difference and the fact, that the number of live born pubs was unaffected by treatment, this effect is considered incidental.

ORGAN WEIGHTS
An increase in absolute (+ app. 18%) and relative (+18.9% males, 19.7% females) liver weight was observed for animals receiving 12000ppm DEHA. No other groupp treatment group was effected. This increase in liver weight has been reported previously and is associated with peroxisome proliferation (Moody and Reddy 1978).

GROSS PATHOLOGY
No treatment related abnormalities, with the possible exception of an accentuated lobular pattern in the liver of two high dose females.

HISTOPATHOLOGY
No treatment related abnormalities were observed. This includes, that no microscopic changes were detected in the reproductive tract of animals which failed to breed successfully, to account for suspected infertility.
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
ca. 1 080 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no effects observed
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
ca. 170 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: increased absolute liver weights (males and females), and reduced body weight gain in females
VIABILITY (OFFSPRING)
There were 4 whole litter losses. None in the control group, one in the 300ppm DEHA dose group, two in the 1800ppm DEHA dose group, and one
in the 12000ppm DEHA dose group. These were of a low lncidence, not dose related, and therefore not related to treatment with DEHA.

CLINICAL SIGNS (OFFSPRING)
No treatment related effect

BODY WEIGHT
There was no difference in pub mean weight on PND1, but mean pup weight gain and consequently total litter weight for high dose male and female offspring were reduced throughout the whole of the post partum phase. There was no effect on either male or female pup weight gain in any other dose group in comparison with the control animals.

GROSS PATHOLOGY (OFFSPRING)
No treatment related effect
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 170 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Mean pup weight gain and total litter weight for both male and female offspring, supposedly secondary to similar effect in parental animals.
Reproductive effects observed:
no
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 080 mg/kg bw/day
Additional information

The one-generation study (CEFIC, 1988) was considered key, as it was a GLP study performed equivalent to OECD Guideline 415. In this study 15 male and 30 female Wistar rats per group were fed DEHA in the diet from 10 weeks before mating and through mating (males) or until day 22 postpartum (females) at levels of 28, 170 and 1080 mg/kg bw/day (300, 1800 or 12 000 ppm). At 1080 mg/kg, body weight gain was marginally reduced in females during premating. This difference was statistically significant during gestation weeks two and three and led to significant lower body weight (-6%) at the end of gestation. No body weight was reported for the lactation period. Liver weights of both male and female parental animals were significantly increased, most likely due to peroxisome proliferation as has already been reported in the literature (Moody D E, Reddy J K (1978). Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds. Toxicol Appl Pharmacol 45 (2) 497-504). Thus, this effect is of no relevance for humans.


There were no effects on male or female fertility, gestation length, and pre-coital interval up to the highest dose level (1080 mg/kg). In the high-dose group, reduced total litter weights, and reduced body weight gain in the pups were observed. However, these effects were most likely secondary to the reduced body weight gain during gestation in the parental animals. There was also a minimal and not statistically significant decrease in litter size in this group, but since there was no difference in the total number of pups born and pup survival, this observation was considered incidental and not toxicologically relevant. No differences in clinical signs or treatment-related macroscopic abnormalities were found in the pups of all groups.


The systemic NOAEL for both generations was conservatively set to 170 mg/kg because of the reduced body weight and / or body weight gain in parental females and the increase in parental liver weights in high dose animals observed at the next higher dose of 1080 mg/kg. Since reproductive performance was unaffected by treatment, the NOAEL for fertility is 1080 mg/kg.


 


In a 28 -day repeated dose study by Miyata et al. (2006, RSS included in IUCLID Section 7.5 Repeated dose toxicity) test substance administration had no effect on sperm parameters, hormonal status, and histopathology of reproductive organs up to 1000 mg/kg with the exception of 4 of 10 high dose females, which showed increased ovarian follicle atresia. Two of these females had a prolonged estrous cycle (4 and 10 days, until sacrifice), although no changes in hormones were detected. In the above cited one-generation study, more than 90% of 30 females treated for 10 weeks with up to 1080 mg/kg became pregnant within 5 days, and no difference in the pre-coital interval was observed compared to control animals. In this study as well as in a chronic study (NTP, 1982) using 50 animals per sex and group at doses up to 1250mg/kg no increase in ovarian follicle atresia was reported as well as no changes in further reproductive organs (Cervix, Epididymis, Mammary gland, Ovary, Prostate, Seminal vesicle, Testis, Uterus). Thus, since the effects observed with only 10 animals after 4 weeks could not be confirmed by two studies using higher animal numbers, which were treated with comparable doses for significantly longer time periods, it is concluded that DEHA does not adversely affect fertility.


 


No hints for estrogenic activity of DEHA were detected in an immature type uterotrophic assay performed by the Korean Food and Drug Administration (Park et al., 2007, see IUCLID section 7.9.3). No changes in absolute organ weights, serum FSH and LH levels, or uterine morphological changes such as luminal epithelial height, myometrial thickness, numbers of uterine glands and BrDU indices were observed up to a dose of 1000mg/kg/day. Furthermore, DEHA was negative in ER- and Androgen-receptor bindung assays (Ghisari et al 2009, ter Veld 2008 and Krüger 2008, see IUCLID section 7.9.3). In addition, there was no biologically meaningful activity in any of the in vitro endocrine disruption screening assays conducted by US EPA (EDSP21 program).


 


The publication by Dalgaard et al. (Reprod. Tox., 2003) was disregarded for the assessment, because the data presented is unreliable. In this non-GLP study, DEHA was fed by gavage to 20 pregnant Wistar rats per group at doses of 200, 400, and 800mg/kg from GD7 to day 17 after parturition. Data on pups are presented for days 3, 13, and 21, but it is unclear, if pups were directly exposed to DEHA or only indirectly via lactation. According to the authors the study design was similar to an OECD 426 study, but there are no similarities between the publication and the OECD 426 guideline. For example, there was no assessment of gross neurologic and behavioural abnormalities, including the assessment of physical development, behavioural ontogeny, motor activity, motor and sensory function, and learning and memory. Instead, additional parameters were included to detect effects on male sexual maturation.


No antiandrogenic effects were found, but the authors state that they observed prolonged gestation time and an increase in postnatal death and decreased body weight in the pubs in the absence of maternal toxicity. These results are questionable due to some severe flaws in the study.


• The assessment of maternal toxicity is not possible based on the data presented, i.e., absence of clinical signs. No further data on hematology, clinical chemistry, or histopathology are available.


• There is limited data on maternal body weight gain from GD7 – 21 showing no significant difference between groups, but dams were not randomly assigned to the treatment groups, but based on their body weight. An evaluation of the data on body weight gain is not possible without individual animal data prior to treatment. Since it is unknown, if this led to selection of pregnant animals for the high dose that were significantly lighter or heavier than control animals, further effects on reproductive parameters cannot be excluded.


• The acclimatization period was very short (4 days, starting on GD3) considering a full reversal of the dark-light cycle and the use of pregnant animals.


• Pup weights cannot be compared between groups. Pups were all weighed on the same day, independent of their age. Postnatal day 0 was defined as gestation day 21, even if pups were not yet born. On average, pups were born 0.9 days later in the high dose group compared to control animals, so that weight differences might solely be due to difference in age.


• The increase in postnatal deaths was slight and not dose dependent – 400mg/kg led to a higher percentage of postnatal deaths than 800mg/kg. Additionally, the percentage of postnatal deaths at 800mg/kg in the pre-study was no different from the controls. The same is true for the stated increase of perinatal deaths. This is not surprising, because of the unusual definition of perinatal death in the publication, which includes all postnatal deaths until day 21.


• No historical control data or data on individual animals are available, making it impossible to judge the biological significance of the differences.


 


In conclusion, many effects observed in the study might be due to poor study design (e.g., animal assignment and data acquisition). An assessment of the remaining differences is impossible, since key information is missing. For all these reasons and because these effects were not observed at a higher dose in a guideline conform one-generation GLP study (CEFIC 1988), the publication presented above is considered invalid.


 


 


The Publication of Wato et al. (2009) was also disregarded due to major methodological defiencies.


The results presented are at least doubtful. For example, the statistics applied are very questionable. The authors state in the fertility study a "significant increase in mean estrous cycle length was observed". But looking at the numbers presented in Table 7 the control groups' value is 4.6 ± 1.7, which is clearly in the range of 4.8 ± 0.5 and 5.4 ± 1.1 respectively (1000 and 2000 mg/kg bw group). The same applies for the number of live embryos (12.0 ± 3.9 compared to 15.4 ± 1.5). Also the authors stated there was "a significant increase in post-implantation loss". However, the mean numbers presented in the table do have a comparably high standard deviation in the dose groups (1.3 ± 2.7 for the control group, 4.8 ± 6.0, 7.6 ± 7.7 and 7.8 ± 4.0 for the dose groups). This is not discussed anywhere. A possible explanation would be for example outliers. Together with the missing historical control data a scientifical judgement on these numbers presented is very debatable.


The lack of historical control data is also a major problem in correctly assessing the histopathological results. For example the authors state "Decrease in currently formed corpus luteum [...] was observed in the 1,000 mg/kg and above". The numbers indicated in the table for the 4 weeks for the 1000 mg/kg bw day study are 3, 2 and 2 (right, left and whole) and for the control group 0, 0 and 0. But in the table for the 2 weeks study the numbers for the control group are 3, 2 and 2. The exact same values as the 1000 mg/kg bw group in the 4 weeks study. Therefore, there is absolutely no indication for an "unnormal" decrease in currently formed corpus luteum. When it comes to the high dose group (2000 mg/kg) it can't be denied, that there are seemingly high values for several observations, even without historical control data. But it must be considered, in the high dose group, a significant decrease in body weight was observed compared with the control group. However, in the food consumption measurement, there were no abnormal changes in both 2 weeks and 4 weeks study. Therefore, the effects observed in the high dose group concerning fertiliy are most probably induced by maternal toxicity. Moreover, the high dose (2000 mg/kg bw) is above the limit dose for repeated dose testing (1000 mg/kg bw). Additionally, there is no information on the purity and identity of the test substance given and there were only 10 animals used per dose.


Taken altogether, the study was considered to be unreliable at least up to the limit dose of 1000 mg/kg bw.


 


Recently, similarities between DEHA and DEHP have been proposed. But contrary to the effects seen with DEHP, no tubular atrophy of the testes or sertoli cell vacuolization have been observed in three repeated dose studies (28 - days, 90 -days, 2 -years), in which rats and mice were exposed to app. 1000 mg/kg. Furthermore, no histopahtological differences in the reproductive organs were observed in the pups of the one-generations study (CEFIC 1988) 22 days postpartum. With DEHP, effects in repeated dose studies using adults animals were detectable after 28 days and pronounced after 60 days of exposure to app. 280 mg/kg DEHP. If pregnant rats were treated with 750 mg/kg DEHP from gestation day 14, decreased male reproductive organ weights, nipples in males, and malformed reproductive organs were already observed at PND 3.


There is also no structural similarity between DEHA and DEHP with the exception of one common metabolite. DEHA is rapidly hydrolysed to adipic acid and ethylhexanol (2 -EH) without accumulation of monoethyl-hexyl-adipate (MEHA). In rats and mice, 2-EH is further metabolised and mainly excreted via urine as glucuronidated ethylhexanoic acid (2-EHA) . In monkeys and humans, (glucuronidated) EHA plays only a minor role (see chapter on toxicokinetics for details). DEHP is also hydrolized in a first step to yield 2 -EH, but further hydrolysis of the resulting monoester (MEHP) is blocked by the adjacent carboxy-group. Instead, MEHP is hydroxylated at different positions within the ethylhexyl chain and eventually glucoronidated and excreted via the kidneys. In 1986 Sjörberg et al. compared the effects of 2 -EH, DEHP, MEHP and three MEHP metabolites in vivo and in vitro (see section 7.9.3). All substaces were administered to rats at 2.7 mmol/kg bw for 5 consecutive days. No testicular damage was seen in rats dosed with 2 -EH, whereas the number of degenerated cells in the seminiferous tubules was increased after exposure to MEHP. This was supported by the observation that MEHP significantly increased the detachment of germ cells in primary rat testicular cell cultures in the range of 1 -200µM. It was thus concluded that MEHP and not 2 -EH is responsible for the testicular damage observed after DEHP exposure.


 


In conclusion, based on all available data, there is no reason to suspect that DEHA impairs fertility.


 


 

Effects on developmental toxicity

Description of key information
Rat, diet: NOAEL teratogenicity = 1080 mg/kg, NOAEL maternal tox. = 170mg/kg (CEFIC 1988, similar to OECD 414, treatment from gestation day 1 to 22, GLP)
Rabbit, diet: NOAEL teratognicity and maternal tox. = 145mg/kg (BASF 2014, OECD 414, GLP)
One-generation reproduction study in rats: reduced pub weights at 1080mg/kg, NOAEL 170mg/kg (CEFIC, 1988, comparable to OECD guideline 415)
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 080 mg/kg bw/day
Additional information

Two prenatal developmental toxicity studies were performed with DEHA in rats and rabbits.


In a study according to GLP and OECD 414 pregnant rabbits were treated with DEHA in the diet at 40, 80 and 160 mg/kg bw/day, which corresponded to 36, 70, and 145mg/kg/day based on the average relative food consumption (BASF, 2014). Dose levels were based on a maternal toxicity range finding study. Severe toxicity was noted at 300 mg/kg bw/day and 1000 mg/kg bw/day. No toxicity was noted at 100 mg/kg bw/day.
The mean test article intake was highest at the beginning and exceeded the average value of 145 mg/kg/day until day 18, which is the most sensitive period in fetal development in rabbits. No maternal toxicity was observed in all dose groups. 20 control and 19 litters per treatment group were available for evaluation. There was no difference between treated and control animals with regard to implantation loss, corpora lutea, litter size, sex distribution, fetal body weight, and external and visceral malformations or variations. Doses were based on a preliminary range finding study, in which 5 mated females were exposed to 100, 300, and 1000 mg/kg from days 7 -29 post coitum. Severe toxicity (e.g., body weight loss) was noted after treatment with 300 mg/kg in the diet and via gavage (to exclude palatability problems), so that the maximum dose in the main study was app. one half of 300 mg/kg.


Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) was established as being at least 160mg/kg bw/day in the diet (dose level approximation). Based on the average relative food consumption, this level corresponded to 145 mg/kg bw/day.


 


24 pregnant Wistar rats were exposed via the diet to on average 28, 170, and 1080mg/kg from gestation day 1 to 22 in a study following GLP criteria and similar to OECD guideline 414 (CEFIC, 1988). Administration of 1080 mg/kg resulted in slight maternal toxicity, expressed as a significant reduction in body weight gain and a slight, but also significant reduction in food consumption. There was no effect at any dose on foetal weight, litter weight, gravid uterus weight, numbers of intra-uterine deaths or numbers of external abnormalities. The incidence of minor external and visceral defects was unaffected by treatment although two visceral variants were increased at the top two dose levels, kinked ureter being increased in the 1800 and 12000 ppm groups, though not statistically significant, and slightly dilated ureter being increased in the 12000 ppm group. Incidences for both effects were within historical control data and are thus not considered adverse. Findings indicate dose-dependent slightly poorer ossification at the 1800 and 12000 ppm dose levels. The reduced ossification are not considered adverse, but as the result of minimal foetotoxicity.


 


In a supporting publication by Singh et al only 5 rats per group were treated by intraperitoneal injection, with up to 10 ml/kg (9250 mg/kg b.w.). No increase in skeletal malformations, visceral or gross abnormalities and no differences in the number of corpora lutea, resorptions, and live fetuses were observed. The two highest doses (5 and 10 ml/kg) led to a slight but significant reduction in foetal body weight, but the relevance of this effect cannot be assessed, since no data on maternal toxicity was presented.


 



Justification for selection of Effect on developmental toxicity: via oral route:
In two OECD 414 studies in rats and rabbits, no adverse effects on the offspring were observed up to the highest dose-tested. Consequently, the NOAEL for developmental toxicity was set to 1080 mg/kg bw.

Justification for classification or non-classification

The available developmental and one-generation reproduction toxicity data do not trigger classification for developmental toxicity according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008).

Additional information

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