Registration Dossier

Diss Factsheets

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.

Reproduction data are summarised in Table 1.
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).
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
No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).
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

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
  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% (**)

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

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

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% -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).

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
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

Constituent 1
Chemical structure
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
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
  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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