Registration Dossier
Registration Dossier
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EC number: 217-288-0 | CAS number: 1800-91-5
Developmental toxicity / teratogenicity
Administrative data
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 15-JAN-2019 to 19-FEB-2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 15-JAN-2019 to 19-FEB-2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D-A19 -09_03_2018
- Expiration date of the lot/batch: 31 October 2023
- Purity test date: 19 October 2018 - Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Crl: WI(Han), outbred, SPF-quality
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: 9-10 weeks old; Females: 12-14 weeks old
- Weight at study initiation: Males: 243 to 270 g ; Females: 177 and 236 g
- Fasting period before study: No
- Housing: Polycarbonate cages (Macrolon, MIV type, height 18 cm).
During pre-mating period up to 5 animals of the same sex and same dosing group together.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Males: 6 days before the commencement of dosing; Females: 5 days prior to start of the pretest period.
DETAILS OF FOOD AND WATER QUALITY:
- Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Municipal tap water was freely available to each animal via water bottles. Periodic analysis of the water was performed.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target 18 to 24°C; actual mean temperature: 19 to 21°C
- Humidity (%): Target: 40 to 70% ; actual daily mean : 48 to 75%
- Air changes (per hr): 10 or more ACH
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
IN-LIFE DATES: From: 07-MAY-2019 To: 09-JUL-2019 - Route of administration:
- oral: gavage
- Vehicle:
- other: No vehicle used to avoid homogeneity issue in aqueous vehicle
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
No vehicle was used.
Dose volumes for group 2 to 4 were calculated as dose level (g/kg) / density:
group 1 (water): 0.637 ml/kg
group 2 (50 mg/kg/day): 0.032 ml/kg
group 3 (250 mg/kg/day): 0.159 ml/kg
group 4 (1000 mg/kg/day): 0.637 ml/kg
The control group animals received water at a dose-volume similar to the high dose group. - Details on mating procedure:
- - M/F ratio per cage: After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating.
- Length of cohabitation: until successful mating, or a maximum of 14 days.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear, referred to as day 0 post-coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility. In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a male for a maximum of 7 days (if possible). A male of the same group having previously shown evidence of mating (non-selected male if possible) will be used for re-mating
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged individually.
- Any other deviations from standard protocol: no - Details on analytical verification of doses or concentrations:
- No vehicle was used for the administration due to observed lack of homogeneity in aqueous vehicles.
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures - Duration of treatment / exposure:
- Males: 29 days
females: 50-64 days
Females which failed to deliver or had a total litter loss were treated for 41-43 days. - Frequency of treatment:
- Daily administration
- Details on study schedule:
- - Age at mating of the mated animals in the study: mating started after a 14-day dosing period.
males:11-12 weeks ; females 14-16 weeks - Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 250 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes
- Details on study design:
- - Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral gavage administration
- Rationale for animal assignment: computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.
- Fasting period before blood sampling for clinical biochemistry: Yes, F0-males were fasted overnight with a maximum of 24 hours ; F0- females were not fasted overnight. - Positive control:
- Not required
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for general health conditions/mortality, in the morning and at the end of the workday.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first dosing, then once weekly throuhout treatment. The observations
were conducted after dosing.
BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing, then weekly
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly, except for males and females housed together for mating, and females without evidence of mating.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- no quantitative investigation done for this study, only visual check.
OTHER: see repeated dose toxicity section - Oestrous cyclicity (parental animals):
- Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. - Sperm parameters (parental animals):
- Not performed
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals at PND 14-16
GROSS NECROPSY
- Gross necropsy described in section 7.5.1 (RDT section)
HISTOPATHOLOGY / ORGAN WEIGHTS
Details reported in described in section 7.5.1 (RDT section) - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring were sacrificed at PND 14-16.
- These animals were subjected to postmortem examinations (macroscopic examination) :
Pups that died before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated
GROSS NECROPSY
Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
HISTOPATHOLOGY / ORGAN WEIGTHS: not performed in this study - Statistics:
- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Parametric tests:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Non-parametric tests:
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant. - Reproductive indices:
- male number paired with, mating date, confirmation of pregnancy and delivery day.
Mating index, precoital time, fertility index, gestation index, duration of gestation - Offspring viability indices:
- Post-implantation survival index, live birth index; % live males at first litter check, % live females at first litter check, viability index, lactation index
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- The statistically significantly increased relative food consumption in females at 1000 mg/kg/day over post coitum Days 17-20 (109% of control) was considered to be the result of a slightly low control value. As all values remained within the normal range, this slight difference was considered not toxicologically relevant.
Historical control data for Wistar Han rats; F0-females (period 2017-2019):
Relative food consumption (g/kg bw/dy) - Day 19 post coitum: mean = 70; P5-P95 = 55.1 – 89.6 (n=462). - Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- no quantitative records, only visual check.
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- No effects on haematology parameters or coagulation parameters up to 1000 mg/kg/d.
Activated partial thromboplastin time (APTT) was shorter in both males (0.80x) and females (0.83x) at 1000 mg/kg/day. The differences were not statistically significant and as mean values remained within the historical control data , this change was considered not toxicologically relevant.
Any statistically significant changes in coagulation parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
------------------------------------
Historical control data for APTT in Wistar Han rats; F0-males (period 2017-2019):
Activated partial thromboplastin time (s): mean = 18.0; P5-P95 = 12.9-22.8 (males, n=257).
Historical control data for Wistar Han rats; F0-females (period 2017-2018):
Activated partial thromboplastin time (s): mean = 17.6; P5-P95 = 13.1-22.2 (females, n=203). - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- - Alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activity was relatively high in one female at 1000 mg/kg/day (No. 71), with values being 2.0x and 1.4x of mean control values, respectively. Means did not reach statistical significance. Values of this female were just outside the available historical control range.
There was a slight increase in the mean values in males, dose-dependent, but not reaching statistical significance.
- statistically significant decrease in total bilirubin in males at 1000 mg/kg/day (0.76x). Mean value was at the lower limit of the historical control range.
- Decreased triglycerides in males at 250 (0.89x) and 1000 mg/kg/day (0.84x); not statistically significant and all values were within the control range.
- Decreased bile acids in males at 250 (0.74x) and 1000 mg/kg/day (0.73x); not statistically significant. Values remained within the concurrent control and available historical control range.
-------------------------
Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
- Alanine aminotransferase (U/L): mean = 99.7; P5-P95 = 49.3-144.7 (females, n=145).
- Aspartate aminotransferase (U/L): mean = 99.7; P5-P95 = 75.7-129.6 (females, n=145).
- Total bilirubin (μmol/L): mean = 2.3; P5-P95 = 1.60-3.00 (males, n=270).
- Bile acids (μmol/L): mean = 25.6, P5-P95:9.70-53.70 (n=264). - Behaviour (functional findings):
- effects observed, non-treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings after treatment with 1,6-divinylperfluorohexane were noted in the liver of males and females and the thyroid gland of females. (see RDT summary for details)
LIVER: hepatocellular vacuolation centrilobular macro and micro vesicular was present in all males at 50, 250 and 1000 mg/kg/day up to marked degree and in a single (1/5) female at 1000 mg/kg/day at slight degree.
This finding correlated with the macroscopically enlarged liver in males and females at 1000 mg/kg/day and, for males, this also correlated with the increased liver weight at 250 and 1000 mg/kg/day.
THYROID: follicular cell hypertrophy was present at increased incidence and/or severity in 4/5 females at 250 and in 5/5 females at 1000 mg/kg/day up to slight degree. (Table 4)
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. - Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Thyroid hormone analyses (Table 5):
- F0-males : Serum levels of T4 in were considered unaffected by treatment up to 1000 mg/kg/day.
At 250 and 1000 mg/kg/day, serum T4 levels were slightly lower than concurrent control (not statistically significant), but values remained within the historical control range and were therefore considered to be of no toxicological significance.
Historical control data for Wistar Han rats; F0-males (period 2017-2019):
Total T4 (μg/dL): mean = 4.51; P5-P95 = 2.85-6.37 (males, n=557). - Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for one female at 50 mg/kg/day (No. 59) and one female at 1000 mg/kg/day (No. 76). Both females had a normal litter. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment. - Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- * The mating index was 100% for all groups.
* The number of implantation sites was statistically significantly lower at 50 and 1000 mg/kg/day (mean of 11.1 and 10.1 implantation sites, respectively, versus 13.7 in controls).
At 1000 mg/kg/day, this lower mean value was caused by female No. 80 that had a single implantation site only (with no pups). All other individual values remained within the historical control range .
In the absence of a dose-relationship and as all other values remained within the normal range, this single occurrence of one implantation site only was considered to be a chance finding and unrelated to treatment with the test item.
- Historical control data for Wistar Han rats; F0-females (period 2015-2019): Implantation sites: mean = 12.3; P5-P95 = 6.0-16.0 (n=1109).
* The fertility indices were 100, 90, 100 and 80% for the control, 50, 250 and 1000 mg/kg/day groups, respectively. All values were within normal limits .
One female at 50 mg/kg/day (No. 57) and two females at 1000 mg/kg/day (Nos. 75 and 78) were not pregnant.
* The gestation index at 1000 mg/kg/day was 88% compared to 100% in the other groups. This was caused by one female (No. 80) that had one implantation site only. All other pregnant females had live offspring.
Duration of gestation was considered not to be affected by treatment with the test item.
* Parturition/Maternal Care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
* Post-Implantation Survival Index
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 92, 98 and 93% for the control, 50, 250 and 1000 mg/kg/day groups, respectively.
For one female (No.58, 50 mg/kg/day), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- organ weights and organ / body weight ratios
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs occurred among pups that were considered to be related to treatment with the test item.
The nature and incidence of clinical signs noted remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant. - Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- Litter data reported in Table 2
Litter size was considered not affected by treatment with the test item.
A statistically significantly lower mean number of living pups was recorded at 1000 mg/kg/day (10.7 vs. 12.5 in the control group). As a dose-related response was absent and all individual values remained both within the concurrent control range and the range considered normal for rats of this strain and age, this difference was considered not to be toxicologically relevant.
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment with the test item. The live birth indices were 97% for the control and 100% for the 50, 250 and 1000 mg/kg/day groups.
Four pups of the control group were found dead at first litter check. As this considered the control group and the mortality incidence remained within the range considered normal for pups of this age, no toxicological relevance was attributed to these dead pups.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item. Viability index was 100% for the control, 50 and 250 mg/kg/day groups, and 99% at 1000 mg/kg/day.
One pup at 1000 mg/kg/day was found missing on PND 2. This pup was most likely cannibalised. No toxicological relevance was attributed to this missing pup as the mortality incidence remained within the range considered normal for pups of this age.
Lactation index was 100% for all groups. No pups were found dead or missing between lactation days 5 and 13. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Pups body weight reported in Table 3.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- Thyroid hormone (T4 levels): Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item. (Table 6)
- Anogenital distance (AGD):
- no effects observed
- Description (incidence and severity):
- Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.
- Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment. - Other effects:
- no effects observed
- Description (incidence and severity):
- Sex ratio was considered not to be affected by treatment with the test item
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- The study did not show toxicologically significant changes in the reproductive parameters investigated in this study (i.e. mating index, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).
No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day): no effects were noted on sex ratio and early postnatal pup development parameters (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)). - Executive summary:
The test substance 1,6-divinylperfluorohexane given orally by gavage for a minimum of 28 days to Wistar Han rats (10 males and 10 females/dose group) was investigated in an OECD422 test guideline study, to assess the potential toxic effects and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.
The dose levels in this study were selected to be 0, 50, 250, 1000 mg/kg/day, based on the results of the Dose Range Finder.
The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males and females) and TSH (F0-females), gross necropsy findings, organ weights and histopathologic examinations.
In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).
Adverse parental findings in this study were present in the liver of males at 1000 mg/kg/day and consisted of hepatocellular vacuolation centrilobular macro and micro vesicular, up to a marked degree. This correlated with the considerable and significant increase in liver weight (both absolute and relative to body weight) and enlargement of the liver. Given the severity of these findings, this was considered adverse.
At the lower dose levels of 50 and 250 mg/kg/day, and in one female at 1000 mg/kg/day, hepatocellular vacuolation was considered not adverse at the lower severity observed. For one female at 1000 mg/kg/day, a slightly higher liver weight and increased liver enzyme activity was noted. However, given the low magnitude of change and in the absence of a histological correlate, these findings were considered to be of no toxicological relevance.
Follicular cell hypertrophy of the thyroid gland of females at 250 and 1000 mg/kg/day was considered non-adverse at the level of severities observed in the current study and in the absence of any degenerative findings. Increased TSH and significantly increased T4 thyroid hormone in females at 1000 mg/kg/day were considered non-adverse, as all values remained within the historical control range.
Minimal changes that were noted in other clinical biochemistry parameters in males at 250 and/or 1000 mg/kg/day (decreased total bilirubin, triglycerides and bile acid levels) were, in the absence of any correlating findings, considered to be of no toxicological relevance.
No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, haematology and coagulation parameters, and male T4 thyroid hormone levels).
At 1000 mg/kg/day, two females were not pregnant and one female had one implantation site only. There were no morphological findings in the reproductive organs or abnormal spermatogenesis that could explain these cases of unsuccessful pregnancy. As all values remained within the normal range and none of the other reproduction parameters were affected, the two non-pregnancies and single occurrence of one implantation site only was were considered to be a chance findings and unrelated to treatment with the test item.
No toxicologically significant changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating index, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).
No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).
In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/ developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for 1,6-divinylperfluorohexane were established:
- Parental NOAEL: 250 mg/kg/day (based on adverse liver findings at 1000 mg/kg/day)
- Reproduction NOAEL: at least 1000 mg/kg/day
- Developmental NOAEL: at least 1000 mg/kg/day
At 1000 mg/kg/day, two females were not pregnant and one female had one implantation site only. There were no morphological findings in the reproductive organs or abnormal spermatogenesis that could explain these cases of unsuccessful pregnancy. As all values remained within the normal range and none of the other reproduction parameters were affected, the single occurrence of one implantation site only was considered to be a chance finding and unrelated to treatment with the test item.
No toxicologically significant changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating index, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).
Table 1- Reproduction data - summary
| Doses (mg/kg/day) | control | 50 | 250 | 1000 | |
| Females paired | 10 | 10 | 10 | 10 | |
| Females mated | 10 | 10 | 10 | 10 | |
| Pregnant females | 10 | 9 | 10 | 8 | |
| Females with implantations only | 0 | 0 | 0 | 1 | |
| Females with living pups on day 1 | 10 | 9 | 10 | 7 | |
| Mating index (%) | 100 | 100 | 100 | 100 | |
| Fertility index (%) | 100 | 90 | 100 | 80 | HCD: mean = 95, P5-P95 = 80 -120 (n=120) |
| Gestation index (%) | 100 | 100 | 100 | 88 | |
| Median precoital time (day) | 2 | 2 | 2 | 3 | |
| Mean precoital time (day) | 3.0 | 2.0 | 1.9 | 2.8 | |
Implantation sites at necropsy (mean) SD |
13.7 1.8 |
11.1 + 2.6 |
12.2 1.9 |
10.1 ++ 3.9 |
Steel-test significant at 5% (+) or 1% (++) |
| Number of uterine implantation sites | 137 | 100 | 122 | 81 |
Table 2 - Litter data - summary
Doses (mg/kg/day) |
control | 50 | 250 | 1000 | |
| Litter number | 10 | 9 | 10 | 7 | |
| Mean duration of gestation (day) | 21.3 | 21.1 | 21.1 | 21.1 | |
| Dead pups at first litter check | |||||
| litter affected | 3 | 0 | 0 | 0 | |
| total number of dead pups | 4 | 0 | 0 | 0 | |
| mean | 0.4 | 0.0 | 0.0 | 0.0 | |
| Living pups at first litter check | |||||
| % of males/females | 50/50 | 49/51 | 49/51 | 45/55 | |
| total | 125 | 92 | 119 | 75 | |
mean SD |
12.5 2.0 |
10.2 2.5 |
11.9 2.0 |
10.7+ 1.1 |
Steel-test significant at 5% (+) or 1% (++) |
| Postnatal loss (day 0 -4) | |||||
| % living pups | 0.0 | 0.0 | 0.0 | 1.3 | |
| Litters affected (number) | 0 | 0 | 0 | 1 | |
| Number of pups lost | 0 | 0 | 0 | 1 | |
| Culled pups | 45 | 22 | 39 | 18 | |
| Living pups on day 4 postpartum | 80 | 70 | 80 | 56 | (8 pups per litter after culling) |
| Mean per litter | 8.0 | 7.8 | 8.0 | 8.0 | |
| Living pups on day 13 postpartum | |||||
| % males/females | 49/51 | 50/50 | 49/51 | 48/52 | |
| total | 80 | 70 | 80 | 56 | |
| mean per litter | 8.0 | 7.8 | 8.0 | 8.0 | |
| Total number of offspring born | 129 | 92 | 119 | 75 | |
| Total number of uterine implantation sites | 137 | 100 | 122 | 81 | |
| Number of live offspring on day 1 after littering | 125 | 92 | 119 | 75 | |
| Number of live offspring on Day 4 before culling | 125 | 92 | 119 | 74 | |
| Number of live offspring on Day 4 after culling | 80 | 70 | 80 | 56 | |
| Number of live offspring on Day 13 after littering | 80 | 70 | 80 | 56 | |
| Post-implantation survival index (%) | 94 | 92 | 98 | 93 | |
| Live birth index (%) | 97 | 100 | 100 | 100 | |
| Viability index (%) | 100 | 100 | 100 | 99 | |
| Lactation index (%) | 100 | 100 | 100 | 100 | |
Table 3 - pups body weights (grams)
| Day | Sex |
|
Group 1 Control |
Group 2 50 mg/kg/day |
Group 3 250 mg/kg/day |
Group 4 1000 mg/kg/day |
| Number of litters | 10 | 9 | 10 | 7 | ||
| 1 | M | Mean bw+SD | 5.9 + 0.5 | 6.3 + 0.4 | 6.0 + 0.4 | 6.2 + 0.8 |
| F | Mean bw+SD | 5.6 + 0.4 | 5.9 + 0.5 | 5.7 + 0.4 | 6.0 + 0.5 | |
| M+F | Mean bw+SD | 5.8 + 0.4 | 6.1 + 0.4 | 5.8 + 0.4 | 6.1 + 0.5 | |
| 4 | M | Mean bw+SD | 8.7 +1.1 | 9.3 + 1.0 | 8.8 + 0.6 | 9.3 + 1.4 |
| F | Mean bw+SD | 8.3 + 0.9 | 8.9 + 1.2 | 8.4 + 0.6 | 9.1 + 1.0 | |
| M+F | Mean bw+SD | 8.5 + 0.9 | 9.1 + 1.1 | 8.6 + 0.6 | 9.1 + 1.1 | |
| 7 | M | Mean bw+SD | 14.7 + 1.4 | 15.4 + 1.5 | 14.6 + 1.1 | 15.4 + 2.0 |
| F | Mean bw+SD | 14.3 + 1.1 | 14.9 + 1.5 | 14.2 + 1.2 | 15.0 + 1.5 | |
| M+F | Mean bw+SD | 14.5 + 1.2 | 15.2 + 1.4 | 14.4 + 1.1 | 15.1 + 1.6 | |
| 13 | M | Mean bw+SD | 28.7 + 1.9 | 29.7 + 2.7 | 28.4 + 2.1 | 29.2 + 2.5 |
| F | Mean bw+SD | 28.0 + 1.5 | 28.9 + 2.6 | 27.8 + 2.3 | 28.8 + 2.4 | |
| M+F | Mean bw+SD | 28.3 + 1.6 | 29.3 + 2.5 | 28.1 + 2.2 | 28.3 + 2.3 |
* Dunnett-test based on pooled variance significant at 5% level or 1% level
Table 4 : Summary of microscopic findings in thyroid gland in parental females
| Females | ||||
| Dose levels (mg/kg/day) | 0 | 50 | 250 | 1000 |
THYROID GLAND number of animals examined |
5 | 5 | 5 | 5 |
| Follicular cell hypertrophy | ||||
| Minimal | 1 | - | 4 | 3 |
| Slight | - | - | - | 2 |
Table 5: summary of T4 and TSH analyses in parental animals
| Males | Females | ||||||||
| Dose levels | mg/kg/day | 0 | 50 | 250 | 1000 | 0 | 50 | 250 | 1000 |
number of animals |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
Total T4 µg/dL |
mean SD |
4.45 0.50 |
4.85 1.23 |
3.78 1.13 |
3.69 0.97 |
2.35 0.27 |
2.30 0.55 |
2.40 0.42 |
2.93* 0.54 |
TSH µIU/mL |
mean SD |
- | - | - | - | 0.240 0.130 |
0.273 0.182 |
0.250 0.134 |
0.341 0.235 |
* Dunnett-test based on pooled variance significant at 5%
Historical control data for Wistar Han rats; female F0-animals (period 2017-2019):
Total T4 (µg/dL): mean = 2.88; P5-P95 = 1.74 - 4.24 (females,n=97).
TSH (µlU/mL): mean = 0.24; P5-P95 = 0.06 - 0.63 (females,n=78).
Table 6: summary of T4 analyses in PND 14-16 pups
| Males | Females | ||||||||
| Dose levels | mg/kg/day | 0 | 50 | 250 | 1000 | 0 | 50 | 250 | 1000 |
number of animals |
N | 10 | 9 | 10 | 7 | 10 | 9 | 10 | 7 |
Total T4 µg/dL |
mean SD |
5.69 0.70 |
5.34 0.41 |
5.52 0.46 |
5.87 0.52 |
5.05 0.79 |
5.08 0.68 |
5.39 0.50 |
5.46 0.60 |
* Dunnett-test based on pooled variance significant at 5% (*) or 1% (**)
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 15-JAN-2019 to 19-FEB-2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D-A19 - 09_03_2018
- Expiration date of the lot/batch: 31 October 2023
- Purity test date: 19 October 2018
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Crl: WI(Han), outbred, SPF-quality
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: 9-10 weeks old; Females: 12-14 weeks old
- Weight at study initiation: Males: 242 to 270 g ; Females: 177 and 236 g
- Fasting period before study: No
- Housing: Polycarbonate cages (Macrolon, MIV type, height 18 cm).
During pre-mating period up to 5 animals of the same sex and same dosing group together.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Males: 6 days before the commencement of dosing; Females: 5 days prior to start of the pretest period.
DETAILS OF FOOD AND WATER QUALITY:
- Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Municipal tap water was freely available to each animal via water bottles. Periodic analysis of the water was performed.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target 18 to 24°C; actual mean temperature: 19 to 21°C
- Humidity (%): Target: 40 to 70% ; actual daily mean : 48 to 75%
- Air changes (per hr): 10 or more ACH
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
IN-LIFE DATES: From: 07-MAY-2019 To: 09-JUL-2019 - Route of administration:
- oral: gavage
- Vehicle:
- other: No vehicle used
- Remarks:
- to avoid homogeneity issue in aqueous vehicle
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
No vehicle was used.
Dose volumes for group 2 to 4 were calculated as dose level (g/kg) / density:
group 1 (water): 0.637 ml/kg
group 2 (50 mg/kg/day): 0.032 ml/kg
group 3 (250 mg/kg/day): 0.159 ml/kg
group 4 (1000 mg/kg/day): 0.637 ml/kg
The control group animals received water at a dose-volume similar to the high dose group. - Details on analytical verification of doses or concentrations:
- No vehicle was used for the administration due to observed lack of homogeneity in aqueous vehicles. The substance was administered directly with a precision syringe.
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures - Duration of treatment / exposure:
- Males: 29 days
females: 50-64 days
Females which failed to deliver or had a total litter loss were treated for 41-43 days. - Frequency of treatment:
- Daily administration (oral gavage)
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- (water)
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 250 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10
- Control animals:
- other: Yes, controls animals received water
- Details on study design:
- - Dose selection rationale:
The dose levels were selected based on the results of a 10-day dose range finder with oral gavage administration at 500 and 1000 mg/kg/day (3 males and 3 females/dose). Animals were examined for clinical signs, mortality, food consumption, body weights on day 1, 5 and 10. At necropsy liver and kidneys were weighed, and examined for histopathological findings.
- Rationale for animal assignment: computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.
- Fasting period before blood sampling for clinical biochemistry: Yes, F0-males were fasted overnight with a maximum of 24 hours ; F0- females were not fasted overnight. - Positive control:
- No, not required
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for general health conditions/mortality, in the morning and at the end of the workday
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first dosing, then once weekly throuhout treatment. The observations were conducted after dosing.
BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing, then weekly
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, weekly, except for males and females housed together for mating, and females without evidence of mating.
WATER CONSUMPTION AND COMPOUND INTAKE:
- no quantitative investigation done for this study
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: samples wollected between 7.00 and 10.30 am.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0 males were faster overnight (max 24 hrs); F0 females were not fasted
- How many animals: 5/sex/group
- Parameters listed below were examined.
COAGULATION PARAMETERS:
- How many animals: 5/sex/group
- Prothrombin Time (PT)
- Activated Partial Thromboplastin Time (APTT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: samples wollected between 7.00 and 10.30 am.
- Animals fasted: F0 males were faster overnight (max 24 hrs); F0 females were not fasted
- How many animals: 5/sex/group
- Parameters listed below were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 for males, and during the last week of lactation (ca PND10-13) for females. FOB was performed after dosing, after completion of the clinical observations.
- Dose groups that were examined: all dose groups, 5 animals/sex/group
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex L/R) / static righting reflex / grip strength (fore- and hind-limb)/ locomotor activity, recorded for 1 hour under normal light conditions.
IMMUNOLOGY: No
OTHER:
Blood samples were processed for serum.
THYROID HORMONES ANALYSIS (SERUM)
- Parental animals: immunoassay for total Thyrosine (T4) analysis for all F0 males and females;
- Pups: 2 pups / litter on PND4, and 2 pups/litter on PND 14-16
THYROID STIMULATING HORMONE (TSH) ANALYSIS (SERUM)
- Parental animals: in all F0 females. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes (list of organs examined in attachment 1)
HISTOPATHOLOGY: Yes, in selected animals (list of organs in attachment 1, except animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue)
In addition, for Males that failed to sire and females that failed to deliver pups: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina - Statistics:
- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Parametric tests:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Non-parametric tests:
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant. - Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- The statistically significantly increased relative food consumption in females at 1000 mg/kg/day over post coitum Days 17-20 (109% of control) was considered to be the result of a slightly low control value. As all values remained within the normal range, this slight difference was considered not toxicologically relevant.
Historical control data for Wistar Han rats; F0-females (period 2017-2019):
Relative food consumption (g/kg bw/dy) - Day 19 post coitum: mean = 70; P5-P95 = 55.1 – 89.6 (n=462). - Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- no quantitative records.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No effects on haematology parameters or coagulation parameters up to 1000 mg/kg/d.
At 1000 mg/kg/day: Activated partial thromboplastin time (APTT) was shorter in both males (0.80x) and females (0.83x). The differences were not statistically significant and as mean values remained within the historical control data , this change was considered not toxicologically relevant.
Any statistically significant changes in coagulation parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
-----------------------------
Historical control data for APTT in Wistar Han rats; F0-males (period 2017-2019):
Activated partial thromboplastin time (s): mean = 18.0; P5-P95 = 12.9-22.8 (males, n=257).
Historical control data for Wistar Han rats; F0-females (period 2017-2018):
Activated partial thromboplastin time (s): mean = 17.6; P5-P95 = 13.1-22.2 (females, n=203). - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Main variations are summarised in Table 1.
- Alanine aminotransferase (ALAT) activity was dose-dependently increased in males. Statistical significance was not reached, values remained well within the historical control range and control values were relatively low. Therefore, this change was considered not toxicologically relevant.
- Alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activity was relatively high in one female at 1000 mg/kg/day (No. 71), with values being 2.0x and 1.4x of mean control values, respectively. Means did not reach statistical significance. Values of this female were just outside the available historical control range.
- statistically significant decrease in total bilirubin in males at 1000 mg/kg/day (0.76x). Mean value was at the lower limit of the historical control range.
- Decreased triglycerides in males at 250 (0.89x) and 1000 mg/kg/day (0.84x); not statistically significant and all values were within the control range.
- Decreased bile acids in males at 250 (0.74x) and 1000 mg/kg/day (0.73x); not statistically significant. Values remained within the concurrent control and available historical control range.
----------------------------------
Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
Alanine aminotransferase (U/L): Males, mean = 50.9; P5-P95 = 32.70 - 75.10 (n = 270) ; Females, mean = 99.7; P5-P95 = 49.3 - 144.7 (n= 145)
Aspartate aminotransferase (U/L): mean = 99.7; P5-P95 = 75.7 - 129.6 (Females, n= 145).
Total bilirubin (µmol/L): mean = 2.3; P5-P95 = 1.60 - 3.00 (males, n= 270).
Bile acids (µmol/L): mean = 25.6, P5-P95: 9.70 - 53.70 (males, n= 264). - Behaviour (functional findings):
- effects observed, non-treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- In males, higher liver weights were noted in the 250 and 1000 mg/kg/day groups, with statistically significant increase in relative liver weight of 16% and 40%, respectively, and a statistically significant increase in the absolute liver weight at 1000 mg/kg/day. (see Table 2)
In females, there was a slight increase in mean weights (absolute or relative) for the high dose group, which did not reach statistical significance.
For one female at 1000 mg/kg/day, the liver weights (both absolute and relative) was just above the upper limit of the control range, but the values remained within the historical control range.
------------------------------
Historical control data for Wistar Han rats; F0-females (period 2017-2018):
Liver weight - absolute (gram): mean = 11.68; P5-P95 = 9.12-14.08 (n=135).
Liver weight – relative to body weight (gram): mean = 4.14; P5-P95 = 3.50-4.81 (n=135).
The relative liver weight in the control group was also slightly above the historical control data mean.
There were no other test item-related organ weight changes. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- At 1000 mg/kg/day: 1/10 males and 1/10 females had an enlarged liver
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings after treatment with 1,6-divinylperfluorohexane were noted in the liver of males and females and the thyroid gland of females.
LIVER (Table 3) (males and females): hepatocellular vacuolation centrilobular macro and micro vesicular was present in all males at 50, 250 and 1000 mg/kg/day up to marked degree and in a single (1/5) female at 1000 mg/kg/day at slight degree.
This finding correlated with the macroscopically enlarged liver in males and females at 1000 mg/kg/day and, for males, this also correlated with the increased liver weight at 250 and 1000 mg/kg/day.
THYROID (Table 5) (females): follicular cell hypertrophy was present at increased incidence and/or severity in 4/5 females at 250 and in 5/5 females at 1000 mg/kg/day up to slight degree.
Follicular cell hypertrophy of the thyroid gland of females at 250 and 1000 mg/kg/day was considered non-adverse at the level of severities observed in the current study and in the absence of any degenerative findings.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. - Other effects:
- no effects observed
- Description (incidence and severity):
- Thyroid hormone (T4) analyses (Table 6):
- F0-males : Serum levels of T4 in were considered unaffected by treatment up to 1000 mg/kg/day.
At 250 and 1000 mg/kg/day, serum T4 levels were slightly lower than concurrent control (not statistically significant), but values remained within the historical control range and were therefore considered to be of no toxicological significance.
F0 females: Serum levels of T4 in F0-females were increased with statistical significance at 1000 mg/kg/day (x1.25). Values remained within the historical control range and therefore this change was considered non-adverse.
There was no changed in thyroid weights (absolute or relative to bw) (Table 4).
---------------------
Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
Total T4 (µg/dL): mean = 4.51; P5-P95 = 2.85 - 6.37 (males, n=557).
Total T4 (µg/dL): mean = 2.88; P5-P95 = 1.74 - 4.24 (females, n=97).
--------------------
Thyroid Stimulating Hormone (TSH) analyses (Table 6):
- F0-females: At 1000 mg/kg/day, serum levels of TSH were higher than concurrent control (1.42x of control, not statistically significant), but as mean values remained within the historical control range this was considered non-adverse
For F0 males, assessment of TSH was considered not relevant because no treatment-related changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded
----------------
Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
TSH (µlU/mL): mean = 0.24; P5-P95 = 0.06 - 0.63 (females, n=78).
---------------- - Details on results:
- Reproduction and developmental data are reported in section 7.8.1 & 7.8.2
Adverse effects following repeated administration:
Adverse parental findings were present in the liver of males at 1000 mg/kg/day and consisted of hepatocellular vacuolation centrilobular macro- and micro-vesicular, up to a marked degree. This marked effect correlated with the substantial and significant increase in liver weight (both absolute, +37%, and relative to body weight, +40%) and enlargement of the liver at the dose 1000 mg/kg/day. Given the severity of these findings, the liver effects were considered adverse (Kerlin et al. 2016, Tox. Pathol 44(2):147-162).
At the lower dose levels of 50 and 250 mg/kg/day, and in one female at 1000 mg/kg/day, hepatocellular vacuolation was considered not adverse at the lower severity observed.
Follicular cell hypertrophy of the thyroid gland of females at 250 and 1000 mg/kg/day was considered non-adverse at the severities observed in the current study and in the absence of any degenerative findings. Increased TSH and significantly increased T4 thyroid hormone in females at 1000 mg/kg/day were considered non-adverse, as all values remained within the historical control range.
Minimal changes that were noted in other clinical biochemistry parameters in males at 250 and/or 1000 mg/kg/day (decreased total bilirubin, triglycerides and bile acid levels) were, in the absence of any correlating findings, considered to be of no toxicological relevance. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- ca. 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- presumably yes
- Conclusions:
- Repeated administration of 1,6-divinylperfluorohexane produced adverse findings in the liver of males at 1000 mg/kg/day which consisted of hepatocellular vacuolation centrilobular macro- and micro-vesicular, up to a marked degree. This correlated with the substantial and significant increase in liver weight (both absolute and relative to body weight) and enlargement of the liver. Given the severity of these findings, they were considered adverse.
At the lower dose levels of 50 and 250 mg/kg/day, and in one female at 1000 mg/kg/day, hepatocellular vacuolation was considered not adverse at the lower severity observed and in the absence of other critical biochemical changes.
The systemic parental NOAEL was established at 250 mg/kg/day, based on adverse liver findings at 1000 mg/kg/day. - Executive summary:
The test substance 1,6-divinylperfluorohexane given orally by gavage for a minimum of 28 days to Wistar Han rats (10 males and 10 females/dose group) was investigated in an OECD422 test guideline study, to assess the potential toxic effects and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.
The dose levels in this study were selected to be 0, 50, 250, 1000 mg/kg/day, based on the results of the Dose Range Finder.
The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males and females) and TSH (F0-females), gross necropsy findings, organ weights and histopathologic examinations.
In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy,
measurement of thyroid hormone T4 (PND 14-16 pups)).
Adverse parental findings in this study were present in the liver of males at 1000 mg/kg/day and consisted of hepatocellular vacuolation centrilobular macro and micro vesicular, up to a marked degree in 3 out of 5 males. This correlated with the substantial and significant increase in liver weight (both absolute, +37%, and relative to body weight, +40%) and enlargement of the liver. Given the severity of these findings, this was considered adverse.
At the lower dose levels of 50 and 250 mg/kg/day, and in one female at 1000 mg/kg/day, hepatocellular vacuolation was considered not adverse at the lower severity observed. For one female at 1000 mg/kg/day, a slightly higher liver weight and increased liver enzyme activity was noted. However, given the low magnitude of change and in the absence of a histological correlate, these findings were considered to be of no toxicological relevance.
Follicular cell hypertrophy of the thyroid gland of females at 250 and 1000 mg/kg/day was considered non-adverse at the level of severities observed in the current study and in the absence of any degenerative findings. Increased TSH and significantly increased T4 thyroid hormone in females at 1000 mg/kg/day were considered non-adverse, as all values remained within the historical control range.
Minimal changes that were noted in other clinical biochemistry parameters in males at 250 and/or 1000 mg/kg/day (decreased total bilirubin, triglycerides and bile acid levels) were, in the absence of any correlating findings, considered to be of no toxicological relevance.
No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, haematology and coagulation parameters, and male T4 thyroid hormone levels).
At 1000 mg/kg/day, two females were not pregnant and one female had one implantation site only. There were no morphological findings in the reproductive organs or abnormal spermatogenesis that could explain these cases of unsuccessful pregnancy. As all values remained within the normal range and none of the other reproduction parameters were affected, the two non-pregnancies and single occurrence of one implantation site only was were considered to be a chance findings and unrelated to treatment with the test item.
No toxicologically significant changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating index, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).
No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).
In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/ developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for 1,6-divinylperfluorohexane were established:
- Parental NOAEL: 250 mg/kg/day (based on adverse liver findings at 1000 mg/kg/day)
- Reproduction NOAEL: at least 1000 mg/kg/day
- Developmental NOAEL: at least 1000 mg/kg/day
Table 1 - clinical biochemistry - main variations
| Males | Females | |||||||
| Dose levels (mg/kg/day) | 0 | 50 | 250 | 1000 | 0 | 50 | 250 | 1000 |
ALAT, mean SD |
44.6 8.4 |
49.4 11.3 |
53.7 14.1 |
56.8 3.1 |
113.4 9.1 |
108.0 15.1 |
115.6 10.7 |
131.8 53.2 |
| difference to control | - | +10% | +20% | +27% | - | -4.8% | +2.0% | +16.2% |
Total bilirubin, mean SD |
2.1 0.5 |
2.0 0.2 |
1.9 0.3 |
1.6* 0.2 |
2.3 0.3 |
2.0 0.3 |
2.3 0.3 |
2.2 0.1 |
| difference to control | - | -4.8% | -9.5% | -23.8% | - | -13.0% | 0 | -4.3% |
Triglycerides, mean SD |
0.75 0.35 |
0.70 0.10 |
0.67 0.24 |
0.63 0.08 |
0.45 0.27 |
0.25 0.12 |
0.25 0.13 |
0.35 0.21 |
| Difference to control | - | -6.7% | -10.7% | -16% | - | -44.4% | -44.4% | -22.2% |
Bile acids, mean SD |
20.1 9.0 |
17.2 7.1 |
14.8 3.7 |
14.7 3.3 |
21.1 14.9 |
16.8 6.8 |
13.0 4.4 |
22.4 12.9 |
| Difference to control | - | -14.4% | -26.4% | -26.9% | - | -20.4% | -38.4% | +6.2% |
*Dunnett-test based on pooled variance significant at 5%
Table 2: Liver weights (absolute and relative) and mean percent differences from control groups
| Males | Females (1) | |||||||
dose levels mg/kg/day |
0 | 50 | 250 | 1000 | 0 | 50 | 250 | 1000 |
| Number of animals | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
Body weight (g) mean SD |
341 22 |
342 24 |
324 25 |
328 23 |
272 18 |
267 16 |
266 22 |
258 23 |
Absolute liver weight (g) mean SD |
8.63 0.84 |
9.12 0.52 |
9.76 0.79 |
11.80** 1.52 |
11.47 0.72 |
11.12 0.80 |
10.74 0.52 |
12.13 0.97 |
% increase compared to controls |
- |
+6 |
+13 |
+37** |
- |
-3 |
-6 |
-6 |
Relative to body weight (%) mean SD |
2.59 0.08 |
2.76 0.12 |
3.01** 0.15 |
3.62** 0.32 |
4.21 0.20 |
4.24 0.25 |
4.23 0.08 |
4.46 0.27 |
% increase compared to controls |
- |
+7 |
+16** |
+40** |
- |
+1 |
0 |
+6 |
**: p < 0.01
(1) Historical control data for Wistar Han rats; F0-females (period 2017-2018):
Liver weight - absolute (gram): mean = 11.68; P5-P95 = 9.12 - 14.08 (n=135).
Liver weight – relative to body weight (gram): mean = 4.14; P5-P95 = 3.50 - 4.81 (n=135).
Table 3: Summary of microscopic findings in liver
|
Males |
Females |
||||||
Dose levels (mg/kg/day) |
0 |
50 |
250 |
1000 |
0 |
50 |
250 |
1000 |
LIVER number of animals examined |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
Grading of vacuolation hepatocellular centrilobular macro- and micro-vesicular |
|
|
|
|
|
|
|
|
grade 1 - Minimal |
- |
1 |
- |
- |
- |
- |
- |
- |
grade 2 - Slight |
- |
3 |
- |
- |
- |
- |
- |
1 |
grade 3 - Moderate |
- |
1 |
5 |
2 |
- |
- |
- |
- |
grade 4 - Marked |
- |
- |
- |
3 |
- |
- |
- |
- |
Total affected |
- |
5 |
5 |
5 |
- |
- |
- |
1 |
Mean grade |
- |
2.0 |
3.0 |
3.6 |
- |
- |
- |
2.0 |
Table 4: Thyroid weights (absolute and relative) in parental animals
|
Males |
Females |
||||||
dose levels mg/kg/day |
0 |
50 |
250 |
1000 |
0 |
50 |
250 |
1000 |
Number of animals |
10 |
10 |
9 |
10 |
10 |
10 |
10 |
10 |
Absolute thyroid weight (g) mean SD |
0.021 0.004 |
0.020 0.004 |
0.020 0.002 |
0.019 0.002 |
0.015 0.003 |
0.015 0.002 |
0.014 0.002 |
0.015 0.002 |
Relative to body weight (%) mean SD |
0.006 0.001 |
0.006 0.001 |
0.006 0.001 |
0.006 0.001 |
0.005 0.001 |
0.006 0.001 |
0.005 0.001 |
0.006 0.001 |
Table 5: Summary of microscopic findings in thyroid gland in females
|
Females |
|||
Dose levels (mg/kg/day) |
0 | 50 | 250 | 1000 |
THYROID GLAND number of animals examined |
5 | 5 | 5 | 5 |
| Follicular cell hypertrophy | ||||
| grade 1 - Minimal | 1 | - | 4 | 3 |
| grade 2 - Slight | - | - | - | 2 |
| Total affected | 1 | - | 4 | 5 |
| Mean grade | 1.0 | - | 1.0 | 1.4 |
Table 6: summary of T4 and TSH analyses
| Males | Females | ||||||||
| Dose levels | mg/kg/day | 0 | 50 | 250 | 1000 | 0 | 50 | 250 | 1000 |
number of animals |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
Total T4 µg/dL |
mean SD |
4.45 0.50 |
4.85 1.23 |
3.78 1.13 |
3.69 0.97 |
2.35 0.27 |
2.30 0.55 |
2.40 0.42 |
2.93* 0.54 |
TSH µIU/mL |
mean SD |
- | - | - | - | 0.240 0.130 |
0.273 0.182 |
0.250 0.134 |
0.341 0.235 |
* Dunnett-test based on pooled variance significant at 5%
- not analysed
Historical control data for Wistar Han rats; female F0-animals (period 2017-2019):
Total T4 (µg/dL): mean = 2.88; P5-P95 = 1.74 - 4.24 (females, n=97).
TSH (µlU/mL): mean = 0.24; P5-P95 = 0.06 - 0.63 (females, n=78).
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2020
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: OECD guideline 422
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- 3,3,4,4,5,5,6,6,7,7,8,8-dodecafluorodeca-1,9-diene
- EC Number:
- 217-288-0
- EC Name:
- 3,3,4,4,5,5,6,6,7,7,8,8-dodecafluorodeca-1,9-diene
- Cas Number:
- 1800-91-5
- Molecular formula:
- C10H6F12
- IUPAC Name:
- 3,3,4,4,5,5,6,6,7,7,8,8-dodecafluorodeca-1,9-diene
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D-A19 -09_03_2018
- Expiration date of the lot/batch: 31 October 2023
- Purity test date: 19 October 2018
Test animals
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl: WI(Han), outbred, SPF-quality
- Details on test animals or test system and environmental conditions:
- Crl: WI(Han), outbred, SPF-qualityTEST ANIMALS
- Source: Charles River Deutschland
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: 9-10 weeks old; Females: 12-14 weeks old
- Weight at study initiation: Males: 243 to 270 g ; Females: 177 and 236 g
- Fasting period before study: No
- Housing: Polycarbonate cages (Macrolon, MIV type, height 18 cm).
During pre-mating period up to 5 animals of the same sex and same dosing group together.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet: ad libitum
- Acclimation period: Males: 6 days before the commencement of dosing; Females: 5 days prior to start of the pretest period.
DETAILS OF FOOD AND WATER QUALITY:
- Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Municipal tap water was freely available to each animal via water bottles. Periodic analysis of the water was performed.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target 18 to 24°C; actual mean temperature: 19 to 21°C
- Humidity (%): Target: 40 to 70% ; actual daily mean : 48 to 75%
- Air changes (per hr): 10 or more ACH
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
IN-LIFE DATES: From: 07-MAY-2019 To: 09-JUL-2019
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- unchanged (no vehicle)
- Remarks:
- No vehicle used to avoid homogeneity issue in aqueous vehicle
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
No vehicle was used.
Dose volumes for group 2 to 4 were calculated as dose level (g/kg) / density:
group 1 (water): 0.637 ml/kg
group 2 (50 mg/kg/day): 0.032 ml/kg
group 3 (250 mg/kg/day): 0.159 ml/kg
group 4 (1000 mg/kg/day): 0.637 ml/kg
The control group animals received water at a dose-volume similar to the high dose group. - Analytical verification of doses or concentrations:
- no
- Details on analytical verification of doses or concentrations:
- No vehicle was used for the administration due to observed lack of homogeneity in aqueous vehicles.
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures - Duration of treatment / exposure:
- Males: 29 days
females: 50-64 days
Females which failed to deliver or had a total litter loss were treated for 41-43 days. - Frequency of treatment:
- Daily administration
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 50 mg/kg bw/day (nominal)
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes
- Details on study design:
- - Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral gavage administration
- Rationale for animal assignment: computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.
Examinations
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for general health conditions/mortality, in the morning and at the end of the workday
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first dosing, then once weekly throuhout treatment. The observations were conducted after dosing.
BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing, then weekly
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, weekly, except for males and females housed together for mating, and females without evidence of mating.
WATER CONSUMPTION AND COMPOUND INTAKE:
- no quantitative investigation done for this study
POST-MORTEM EXAMINATIONS: Yes (see section 7.5.1 and 7.8.1)
- Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Not relevant
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No - Fetal examinations:
- Not relevant
Results and discussion
Results: maternal animals
General toxicity (maternal animals)
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- No effects on haematology parameters or coagulation parameters up to 1000 mg/kg/d.
Females: Activated partial thromboplastin time (APTT) was shorter in females (0.83x) at 1000 mg/kg/day. The differences were not statistically significant and as mean values remained within the historical control data, this change was considered not toxicologically relevant.
Any statistically significant changes in coagulation parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
------------------------------------
Historical control data for for APTT in Wistar Han rats; F0-females (period 2017-2018):
Activated partial thromboplastin time (s): mean = 17.6; P5-P95 = 13.1-22.2 (females, n=203). - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- FEMALES:
- Alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activity was relatively high in one female at 1000 mg/kg/day (No. 71), with values being 2.0x and 1.4x of mean control values, respectively. Means did not reach statistical significance. Values of this female were just outside the available historical control range.
-------------------------
Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
- Alanine aminotransferase (U/L): mean = 99.7; P5-P95 = 49.3-144.7 (females, n=145).
- Aspartate aminotransferase (U/L): mean = 99.7; P5-P95 = 75.7-129.6 (females, n=145). - Behaviour (functional findings):
- effects observed, non-treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- In females, there was a slight increase in mean weights (absolute or relative) for the high dose group, which did not reach statistical significance.
For one female at 1000 mg/kg/day, the liver weights (both absolute and relative) was just above the upper limit of the control range, but the values remained within the historical control range.
Historical control data for Wistar Han rats; F0-females (period 2017-2018):
- Liver weight - absolute (gram): mean = 11.68; P5-P95 = 9.12-14.08 (n=135).
- Liver weight – relative to body weight (gram): mean = 4.14; P5-P95 = 3.50-4.81 (n=135).
The relative liver weight in the control group as also slightly above the historical control data mean.
There were no other test item-related organ weight changes. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- At 1000 mg/kg/day: 1/10 males and 1/10 females had an enlarged liver.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings after treatment with 1,6-divinylperfluorohexane were noted in the liver of males and females and the thyroid gland of females. (see RDT summary for details)
LIVER: hepatocellular vacuolation centrilobular macro and micro vesicular was present in all males at 50, 250 and 1000 mg/kg/day up to marked degree and in a single (1/5) female at 1000 mg/kg/day at slight degree.
This finding correlated with the macroscopically enlarged liver in males and females at 1000 mg/kg/day and, for males, this also correlated with the increased liver weight at 250 and 1000 mg/kg/day.
THYROID: follicular cell hypertrophy was present at increased incidence and/or severity in 4/5 females at 250 and in 5/5 females at 1000 mg/kg/day up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Maternal developmental toxicity
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 92, 98 and 93% for the control, 50, 250 and 1000 mg/kg/day groups, respectively.
For one female (No.58, 50 mg/kg/day), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study. - Total litter losses by resorption:
- no effects observed
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The fertility indices were 100, 90, 100 and 80% for the control, 50, 250 and 1000 mg/kg/day groups, respectively. All values were within normal limits.
One female at 50 mg/kg/day (No. 57) and two females at 1000 mg/kg/day (Nos. 75 and 78) were not pregnant.
Effect levels (maternal animals)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
Maternal abnormalities
- Key result
- Abnormalities:
- no effects observed
Results (fetuses)
- Fetal body weight changes:
- not examined
- Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment with the test item. The live birth indices were 97% for the control and 100% for the 50, 250 and 1000 mg/kg/day groups.
Four pups of the control group were found dead at first litter check. As this considered the control group and the mortality incidence remained within the range considered normal for pups of this age, no toxicological relevance was attributed to these dead pups. - Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- Litter size was considered not affected by treatment with the test item. (Table 1)
A statistically significantly lower mean number of living pups was recorded at 1000 mg/kg/day (10.7 vs. 12.5 in the control group). As a dose-related response was absent and all individual values remained both within the concurrent control range and the range considered normal for rats of this strain and age, this difference was considered not to be toxicologically relevant. - Changes in postnatal survival:
- no effects observed
- Description (incidence and severity):
- Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item. Viability index was 100% for the control, 50 and 250 mg/kg/day groups, and 99% at 1000 mg/kg/day.
One pup at 1000 mg/kg/day was found missing on PND 2. This pup was most likely cannibalised. No toxicological relevance was attributed to this missing pup as the mortality incidence remained within the range considered normal for pups of this age. - External malformations:
- no effects observed
Effect levels (fetuses)
- Key result
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Remarks:
- (post-natal development)
Fetal abnormalities
- Abnormalities:
- not examined
Overall developmental toxicity
- Key result
- Developmental effects observed:
- no
Any other information on results incl. tables
Table 1 - Litter data - summary
Doses (mg/kg/day) |
control | 50 | 250 | 1000 | |
| Litter number | 10 | 9 | 10 | 7 | |
| Mean duration of gestation (day) | 21.3 | 21.1 | 21.1 | 21.1 | |
| Dead pups at first litter check | |||||
| litter affected | 3 | 0 | 0 | 0 | |
| total number of dead pups | 4 | 0 | 0 | 0 | |
| mean | 0.4 | 0.0 | 0.0 | 0.0 | |
| Living pups at first litter check | |||||
| % of males/females | 50/50 | 49/51 | 49/51 | 45/55 | |
| total | 125 | 92 | 119 | 75 | |
mean SD |
12.5 2.0 |
10.2 2.5 |
11.9 2.0 |
10.7+ 1.1 |
Steel-test significant at 5% (+) or 1% (++) |
| Postnatal loss (day 0 -4) | |||||
| % living pups | 0.0 | 0.0 | 0.0 | 1.3 | |
| Litters affected (number) | 0 | 0 | 0 | 1 | |
| Number of pups lost | 0 | 0 | 0 | 1 | |
| Culled pups | 45 | 22 | 39 | 18 | |
| Living pups on day 4 postpartum | 80 | 70 | 80 | 56 | (8 pups per litter after culling) |
| Mean per litter | 8.0 | 7.8 | 8.0 | 8.0 | |
| Living pups on day 13 postpartum | |||||
| % males/females | 49/51 | 50/50 | 49/51 | 48/52 | |
| total | 80 | 70 | 80 | 56 | |
| mean per litter | 8.0 | 7.8 | 8.0 | 8.0 | |
| Total number of offspring born | 129 | 92 | 119 | 75 | |
| Total number of uterine implantation sites | 137 | 100 | 122 | 81 | |
| Number of live offspring on day 1 after littering | 125 | 92 | 119 | 75 | |
| Number of live offspring on Day 4 before culling | 125 | 92 | 119 | 74 | |
| Number of live offspring on Day 4 after culling | 80 | 70 | 80 | 56 | |
| Number of live offspring on Day 13 after littering | 80 | 70 | 80 | 56 | |
| Post-implantation survival index (%) | 94 | 92 | 98 | 93 | |
| Live birth index (%) | 97 | 100 | 100 | 100 | |
| Viability index (%) | 100 | 100 | 100 | 99 | |
| Lactation index (%) | 100 | 100 | 100 | 100 | |
Table 2 - pups body weights (grams)
| Day | Sex |
|
Group 1 Control |
Group 2 50 mg/kg/day |
Group 3 250 mg/kg/day |
Group 4 1000 mg/kg/day |
| Number of litters | 10 | 9 | 10 | 7 | ||
| 1 | M | Mean bw+SD | 5.9+0.5 | 6.3+0.4 | 6.0+0.4 | 6.2+0.8 |
| F | Mean bw+SD | 5.6+0.4 | 5.9+0.5 | 5.7+0.4 | 6.0+0.5 | |
| M+F | Mean bw+SD | 5.8+0.4 | 6.1+0.4 | 5.8+0.4 | 6.1+0.5 | |
| 4 | M | Mean bw+SD | 8.7+1.1 | 9.3+1.0 | 8.8+0.6 | 9.3+1.4 |
| F | Mean bw+SD | 8.3+0.9 | 8.9+1.2 | 8.4+0.6 | 9.1+1.0 | |
| M+F | Mean bw+SD | 8.5+0.9 | 9.1+1.1 | 8.6+0.6 | 9.1 + 1.1 | |
| 7 | M | Mean bw+SD | 14.7+1.4 | 15.4+1.5 | 14.6+1.1 | 15.4+2.0 |
| F | Mean bw+SD | 14.3+1.1 | 14.9+1.5 | 14.2+1.2 | 15.0+1.5 | |
| M+F | Mean bw+SD | 14.5+1.2 | 15.2+1.4 | 14.4+1.1 | 15.1+1.6 | |
| 13 | M | Mean bw+SD | 28.7+1.9 | 29.7+2.7 | 28.4 + 2.1 | 29.2+2.5 |
| F | Mean bw+SD | 28.0+1.5 | 28.9+2.6 | 27.8+2.3 | 28.8+2.4 | |
| M+F | Mean bw+SD | 28.3+1.6 | 29.3+2.5 | 28.1+2.2 | 28.3+2.3 |
* Dunnett-test based on pooled variance significant at 5% level or 1% level
Table 3: summary of T4 analyses in PND 14-16 pups
| Males | Females | ||||||||
| Dose levels | mg/kg/day | 0 | 50 | 250 | 1000 | 0 | 50 | 250 | 1000 |
number of animals |
N | 10 | 9 | 10 | 7 | 10 | 9 | 10 | 7 |
Total T4 µg/dL |
mean SD |
5.69 0.70 |
5.34 0.41 |
5.52 0.46 |
5.87 0.52 |
5.05 0.79 |
5.08 0.68 |
5.39 0.50 |
5.46 0.60 |
* Dunnett-test based on pooled variance significant at 5% (*) or 1% (**)
Applicant's summary and conclusion
- Conclusions:
- The study did not show toxicologically significant changes in the reproductive parameters investigated in this study (i.e. mating index, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).
No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day): no effects were noted on sex ratio and early postnatal pup development parameters (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)). - Executive summary:
The test substance 1,6-divinylperfluorohexane given orally by gavage for a minimum of 28 days to Wistar Han rats (10 males and 10 females/dose group) was investigated in an OECD422 test guideline study, to assess the potential toxic effects and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.
The dose levels in this study were selected to be 0, 50, 250, 1000 mg/kg/day, based on the results of the Dose Range Finder.
The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males and females) and TSH (F0-females), gross necropsy findings, organ weights and histopathologic examinations.
In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).
Adverse parental findings in this study were present in the liver of males at 1000 mg/kg/day and consisted of hepatocellular vacuolation centrilobular macro and micro vesicular, up to a marked degree. This correlated with the considerable and significant increase in liver weight (both absolute and relative to body weight) and enlargement of the liver. Given the severity of these findings, this was considered adverse.
At the lower dose levels of 50 and 250 mg/kg/day, and in one female at 1000 mg/kg/day, hepatocellular vacuolation was considered not adverse at the lower severity observed. For one female at 1000 mg/kg/day, a slightly higher liver weight and increased liver enzyme activity was noted. However, given the low magnitude of change and in the absence of a histological correlate, these findings were considered to be of no toxicological relevance.
Follicular cell hypertrophy of the thyroid gland of females at 250 and 1000 mg/kg/day was considered non-adverse at the level of severities observed in the current study and in the absence of any degenerative findings. Increased TSH and significantly increased T4 thyroid hormone in females at 1000 mg/kg/day were considered non-adverse, as all values remained within the historical control range.
Minimal changes that were noted in other clinical biochemistry parameters in males at 250 and/or 1000 mg/kg/day (decreased total bilirubin, triglycerides and bile acid levels) were, in the absence of any correlating findings, considered to be of no toxicological relevance.
No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, haematology and coagulation parameters, and male T4 thyroid hormone levels).
At 1000 mg/kg/day, two females were not pregnant and one female had one implantation site only. There were no morphological findings in the reproductive organs or abnormal spermatogenesis that could explain these cases of unsuccessful pregnancy. As all values remained within the normal range and none of the other reproduction parameters were affected, the two non-pregnancies and single occurrence of one implantation site only was were considered to be a chance findings and unrelated to treatment with the test item.
No toxicologically significant changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating index, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).
No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).
In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/ developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for 1,6-divinylperfluorohexane were established:
- Parental NOAEL: 250 mg/kg/day (based on adverse liver findings at 1000 mg/kg/day)
- Reproduction NOAEL: at least 1000 mg/kg/day
- Developmental NOAEL: at least 1000 mg/kg/day
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