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Administrative data

Description of key information

Results of the OECD 422 screening study (GLP) conducted in Wistar rats administered 1,6-divinylperfluorohexane by the oral route (gavage) at up to 1000 mg/kg/day

- NOAEL: 250 mg/kg/day, based on adverse findings in the liver at 1000 mg/kg/day

The main effects at the highest dose were hepatocellular vacuolation centrilobular macro and micro vesicular, up to a marked degree in males. This correlated with the significant increase in liver weight (both absolute and relative to body weight) and the macroscopic enlargement of the liver. These effects are consistent with an adaptative response.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable studies are available
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Adverse 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 finding 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 at the dose 1000 mg/kg/day (Kerlin et al. 2016, Tox. Pathol 44(2):147-162). No hepatocytic hypertrophy was observed at the dose 1000 mg/kg/day following the administration for 28 days.

Centrilobular vacuolation may indicate fatty changes (e.g., altered lipid metabolism) in the centrilobular hepatocytes. They were of greater severity at the highest dose of 1000 mg/kg/day and thus considered adverse. No other degenerative changes indicative of altered hepatic function were noted, and although increased with a dose trend ALT variations remained limited, these changes can be considered signs of metabolic adaptation.

Due to a large dose-spacing between the mid and high-dose, the NOAEL in the study is 250 mg/kg/day. Dose-related trend in centrilobular vacuolation severity and relative liver weight increase support a No-adverse effect level more likely situated between 250 mg/kg/day and 500 mg/kg/day and dose causing adverse effects situated above 300 mg/kg/day. Therefore the DNEL derived from the starting point of 250 mg/kg/day can be considered conservative.

Justification for classification or non-classification

Based on the results of a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the NOAEL was set at 250 mg/kg/day considering adverse effects occuring in the rat liver at the dose 1000 mg/kg/day.

No hazard classification is considered necessary according to the CLP criteria in EU Regulation (EC) No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

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