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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study period is to be confirmed following confirmation of the testing proposal by ECHA
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
2018
GLP compliance:
yes
Justification for study design:
Refer to 'Any other information on materials and methods incl. tables'

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrahydro-2-methylfuran
EC Number:
202-507-4
EC Name:
Tetrahydro-2-methylfuran
Cas Number:
96-47-9
Molecular formula:
C5H10O
IUPAC Name:
2-methyloxolane
Details on test material:
- Name of test material (as cited in study report): 2-methyltetrahydrofuran; MeTHF CAS 96-47-9
- Substance type: Monoconstituent substance
- Physical state: no information
- Analytical purity: no information

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on analytical verification of doses or concentrations:
Dose formulation analysis was within ± 10% of the nominal concentration.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
625 mg/kg bw/day
No. of animals per sex per dose:
20 male and 20 female

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
In the parental generation there was no treatment-related mortality (other than associated with dystocia in all female groups). There was a dose-related increase in ptyalism that was considered not to be adverse and clinical signs of hypo activity, staggering gait, startle, and tremors in males at 625 mg/kg/day only. There were no effects on bodyweight, food consumption, estrous cycle, mating, fertility, duration of gestation or number of implantation sites.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 625 mg/kg/day, there was a decrease in mean number of live pups on Day 1 p.p. (9.5 vs. 12.1 in controls), associated with a decreased live birth index (76.6% vs. 95.5% in controls) and increased post-implantation loss (20.3% vs. 13.9 in controls). These findings were considered to be test item treatment-related and adverse. At 250 mg/kg/day, there was a lower live birth index (85.8% vs. 95.5% in controls, not statistically significant) that was considered to be test item treatment-related and adverse when compared with Historical Control Data (85.8% vs. 98.6%). When compared to contemporary studies (with control animals that also had difficult deliveries) the value was still below the mean historical index (Mean values = 95.7%; Min = 88.3%; Max= 100%). At 100 mg/kg/day, there were no effects.

Details on results (P0)

There was an increase in the number of females with difficulty to deliver at 625 mg/kg/day. These difficulties resulted in premature euthanasia for human reasons on the day of delivery or soon after and were considered to be test-item treatment related and adverse.
The number of control females with difficulty to deliver exceeded the background range. As a result, a number of investigations were carried out to identify the potential causes, including serology and review of dietary / water analyses. No external confounding factors were identified. During the same period, control females with difficulty to deliver were also observed in contemporaneous studies with delivery phases (i.e. ICH pre- and postnatal development toxicity study and, OECD 421, OECD 422 and OECD 443 toxicity studies). Therefore, these finding were considered to represent evolving background of the Janvier Sprague-Dawley (RjHan:SD) rat strain. It is unclear how this confounding factor may have influenced the study results.

In P generation males there were no effects on mean TSH and T4 plasma levels,

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Sexual maturation:
effects observed, non-treatment-related
Other effects:
effects observed, treatment-related
Description (incidence and severity):
At 625 mg/kg/day, there was a lower mean litter size at birth (11.5 vs. 12.6 in controls) and an increased percentage of cannibalized or found dead pups (71.4% of litter affected vs. 52.6% in controls). At 250 mg/kg/day, there were increased percentages of cannibalized or found dead pups (73.9% of litter affected vs. 52.6% in controls). However, from 250 mg/kg/day onwards, there was an increased incidence of pups with absence of milk into the stomach and low qualitative body temperature, which were considered to represent a lack of maternal care. At 625 mg/kg/day, mean anogenital distance (and normalized mean anogenital distance) were higher. In the absence of any effects on the sexual development of F1 animals, this finding was considered to be non adverse. At 250 and 100 mg/kg/day, there were no effects on sexual development.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 2 pups were allocated to neurobehavioural investigations.
There were no treatment-related deaths, clinical signs, effects on bodyweight or food consumption, neuro startle tests, functional observation battery, motor activity or sexual development in Cohort 2.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 2 pups were allocated to neurobehavioural investigations.
There were no treatment-related deaths, clinical signs, effects on bodyweight or food consumption, neuro startle tests, functional observation battery, motor activity or sexual development in Cohort 2.

Details on results (F1)

No effects on T4 or TSH in plasma samples from non-selected F1 pups nor in T4 or TSH plasma levels in Cohort 1A mlaes or females.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEC
Generation:
F1 (cohort 3)
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
developmental immunotoxicity
Key result
Dose descriptor:
NOAEC
Generation:
F1 (cohort 1A)
Effect level:
625 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
developmental immunotoxicity
Remarks on result:
other: There was no test item treatment-related findings on lymphocyte subtyping at any dose level (625 mg/kg/day was the highest dose level tested).
Key result
Dose descriptor:
NOAEC
Generation:
F1 (cohort 2A)
Effect level:
625 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
developmental neurotoxicity

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

 


Table 40; Female Mortality, Parental Generation























































Dose level
(mg/kg/day)



0



100



250



625



Number of females



24



24



24



24



Decision of euthanasia


(humane grounds)



1 (Day 19 p.c.)



 



 



1 (Day 2 p.p.)


1 (Day 1 p.p.)


litters found dead



Decision of euthanasia (difficulties to deliver)



3 (Day 23 p.c.)



1 (Day 23 p.c.)



1 (Day 3 p.p.)



6 (Day 23 p.c.)


1 (Study Day 99 pregnancy not detected, ~ Day 23 p.c)



Decision of euthanasia


(no delivery, pregnant or non pregnant)



1 (Day 25 p.c.)



3 (Day 26 p.c.)



1 (Day 26 p.c.)



1 (Study Day 109)


2(Day 26 p.c.)



Deaths



5



4



2



12



Final euthanasia



19



20



22



12



 


 


Table 41: Delivery data






























































































Dose level
(mg/kg/day)



0



100



250



625



HCD



Number of pregnant females



23



22



24



21



47



Number of pregnant females with difficulties to deliver (%)



3 (13.0)



1 (4.5)



1 (4.2)



7 (33.3)



0 (0)



Number of females which delivered (%)



19 (82.6)



20 (90.9)



23 (95.8)



14 (66.7)



43 (91.5)



Mean duration of gestation


(days ± SD)



22.0 ± 0.0



22.0 ± 0.0



22.0 ± 0.2



22.2 ± 0.4



22.0 ± 0.5



Mean number of implantation sites (± SD)



14.7 ± 2.6



14.4 ± 2.9



14.8 ± 2.9



14.0 ± 4.0



15.3 ± 2.1



Mean number of live pups on Day 1 p.p. (± SD)



12.1 ± 2.7



11.6 ± 3.4



10.9 ± 2.9



9.5 ± 3.6



12.8 ± 2.4



Mean percent of post-implantation loss (% ± SD)



13.9 ± 9.6



15.1 ± 11.4



13.8 ± 14.0



20.3 ± 20.8



14.7 ± 10.7



Live birth index


(% ± SD)



95.5 ± 7.5



93.7 ± 9.8



85.8 ± 13.1



76.6 ± 28.6



98.6 ± 4.1



Sex ratio on Day 1 p.p.


(% ± SD)



53.7 ± 15.6



50.9 ± 14.6



51.0 ± 15.6



41.4 ± 18.1



48.1 ± 13.8



Sex ratio on Day 21 p.p.


(% ± SD)



51.7 ± 13.7



51.4 ± 12.0



51.8 ± 14.9



44.7 ± 13.6



48.3 ± 9.9



 


Table 42: F1 survival






























































Dose level
(mg/kg/day)



0



100



250



625



HCD



Number of litters



19



20



23



14



43



Number of pups delivered (total)



239



247



296



161



/



Mean litter size at birth (± SD)



12.6 ± 2.5



12.4 ± 3.2



12.9 ± 3.3



11.5 ± 4.3



12.8 ± 2.4



Cannibalized, Nb. pups (%)



4 (1.7)



3 (1.2)



16 (5.4)



14 (8.7)



/



Found dead, Nb. pups (%)



13 (5.4)



15 (6.1)



46 (15.5)



33 (20.5)



/



Litter(s) affected, Nb. (%)



10 (52.6)



9 (45.0)



17 (73.9)



10 (71.4)



/



HCD: Historical Control Data (OECD443 - P generation, 2017-2018, n=2 studies. Nb.: Number


/: not available in HCD. No statistically significant difference vs. controls.


 


Table 43: Mating and fertility data in Cohort 1B






























































Dose level
(mg/kg/day)



0



100



250



625 (a)



HCD



Number of animals paired (M + F)



19+19



20+20



20+20



 



 



Number of females mated



19



20



19



 



19



Female mating index (%)



100.0



100.0



95.0



 



95.0



Mean number of days taken to mate (days ± SD)



2.5 ± 1.7



2.7 ± 1.2



3.3 ± 1.6



 



/



Number of pregnant females



19



18



13*



 



19



Female fertility index (%)



100.0



90.0



68.4



 



100.0



(a): no animals at this dose level, M: Males; F: Females, HCD: Historical Control Data (OECD443 - Cohort 1B, 2017-2018, n = 1 study.


/: not reported, Statistical significance: *: p<0.05.


 


 


Necropsy of the parental males treated at 625 mg/kg/day found an increase in absolute and relative-to-body liver weights (up to -13% in relative-to-body weights; p<0.01). This correlated with hepatocellular hypertrophy at microscopic examination. Increases were also observed in the F1 cohort 1A generation in males and females treated at 625 mg/kg/day (up to +21% in relative-to-body weights of males; p<0.01). These increases correlated with microscopic hepatocellular hypertrophy, although in females the increased final body weights (+9%) could have contributed. There were no other treatment-related organ weight changes. 


 


Table 44: Organ weight changes % vs controls, Parental and F1 generation






























































































































Sex



Male



Female



Dose level (mg/kg/day)



100



250



625



100



250



625



Parental Generation



Number of animals per group



24



23



23



23



23



15



- Final body weight



-1



-3



-2



-3



-2



-4



- Liver


      

.absolute



-1



0



+10**



-7



0



-9



.relative-to-body



-1



+3



+13**



-4



+1



-5



F1 generation



Number of animals per group



20



19



20



20



20



20



- Final body weight



0



-2



-3



+4



+3



+9**



- Liver


      

.absolute



+4



+7



+18**



+6



+8*



+19**



.relative-to-body



+5



+9**



+21**



+2



+5



+10**


        

Statistical significance: **: p<0.01 (the significance concerned the organ weights values and not the percentages).


 


Histopathological changes were restricted to dose-related minimal to slight hepatocellular hypertrophy was noted in males and females treated at ≥100 mg/kg/day in the Parental generation.  This finding was also observed in the F1 generation together with minimal thyroid cell hypertrophy at 625 mg/kg/day. Thyroid cell hypertrophy could be considered as most probably compensatory to the increased hepatic metabolism of thyroid hormones following induction of hepatic microsomal enzymes (Greaves, 2012).


Both liver and thyroid hypertrophy under the conditions of this study were not considered as adverse, but rather adaptive changes.


 


Table 45: Histopathology results Parental and F1  generation


























































































































































Sex



Male



Female



Dose level (mg/kg/day)



0



100



250



625



0



100



250



625



Parental generation



 



 



 



 



 



 



 



 



Number of animals per group



24



24



23



23



20



23



23



15



Liver; hepatocellular hypertrophy



 


   

 


   

. grade 1



-



1



5



14



-



7



6



8



. grade 2



-



-



-



1



-



-



-



1



F1 generation



 



 



 



 



 



 



 



 



Number of animals per group



20



20



19



20



20



20



20



20



Liver, hepatocellular hypertrophy



 


   

 


   

. grade 1



-



5



9



12



-



1



7



11



. grade 2



-



-



-



2



-



-



-



-



Thyroid gland*: follicular cell hypertrophy



 


   

 


   

. grade 1



-



-



1



5



-



-



-



3



-: not observed.


*There were respectively 19, 20, 18 and 20 thyroid glands submitted for microscopic examination in males treated at 0, 100, 250 and 625 mg/kg/day.


 


Neurohistopathological evaluation of the cohort 2 animals found not treatment-related changes in the olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem or cerebellum. There were no effects in the eyes (retina and optic nerve), peripheral nerve, muscle or spinal cord. Morphimetric measurements of the cerebral cortex, hippocampus and cerebellum found no definitive changes related to the test item. There were some statistically significant results, but the study pathologist concluded the following;


Two isolated statistically significant results (higher cerebellum thickness in cohort 2A females treated at 250 or 625 mg/kg/day and lower cornu ammonis thickness in cohort 2B females treated at 250 mg/kg/day) lead to deeper investigations.  It was concluded that these differences were not test item-related in view of the low magnitude of these differences and of the absence of clear dose-relationship. It is noteworthy that spurious statistical “flags” are common in linear measurements from DNT (Developmental NeuroToxicity) studies. 


Based on the integrative weight-of-evidence approach of Garman et al (2016), no evidence of developmental neurotoxicity was concluded because:



  • There was an absence of test item treatment-related brain weight differences, gross abnormalities or microscopic alteration in the central nervous system, either in parents (n=24 per group/sex), 1A (n=20), 2A (n=10) or 2B (n=10) F1 cohorts, either in males or females. No microscopic abnormalities as dysplasia or ectopia were present.

  • There was no evidence of test item treatment-related nervous behavioral effects in females. At in-life examination, ptyalism was observed from 250 mg/kg/day onwards and with dose-related increased incidences. This finding commonly observed after a gavage procedure was considered to be test item treatment-related but not adverse.

  • Both statistically significant differences in linear brain measurements appeared to be inconsistent and/or not directly related to the test item:

  • higher cerebellum thickness in cohort 2A females treated at 625 mg/kg/day at PND 75-90 was inconsistent across brain regions (not seen in any other measured regions), was poorly dose-related across dose-levels, and was inconsistent across sex (not seen in high-dose females from 2B cohort at PND 22).

  • lower cornu ammonis thickness in cohort 2B females treated at 250 mg/kg/day at PND 22 was inconsistent across brain regions (not seen in any other measured regions and specifically not in the gyrus dentatus nor in total hippocampus measurements), was poorly dose-related, and was inconsistent across age (not seen in cohort 2A males or females at PND 75-90; an effect is unlikely when seen only in juvenile but not in adults or, in other words, neurotoxicants should be accentuated in adults).


In conclusion, there was no evidence of developmental neurotoxicity in this study.

Applicant's summary and conclusion

Conclusions:
Systemic toxicity evaluation:
• The No Observed Adverse Effect Levels (NOAELs) for systemic toxicity (excluding
reproductive and developmental toxity endpoints) were considered to be:
• 250 mg/kg/day in males based on clinical signs (hypoactivity, staggering gait, sudden startle and/or tremors) observed at 625 mg/kg/day,
• 625 mg/kg/day in females based on the absence of adverse findings before parturition.

Reproductive/developmental toxicity testing:
Therefore the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 100 mg/kg/day.

Developmental neurotoxicity testing:
Therefore the No Observed Adverse Effect Level (NOAEL) for developmental neurotoxicity was considered to be 250 mg/kg/day.

Developmental immunotoxicity testing:
Therefore the No Observed Adverse Effect Level (NOAEL) for developmental immunotoxicity was considered to be 250 mg/kg/day.

Executive summary:

Systemic toxicity evaluation:
• The No Observed Adverse Effect Levels (NOAELs) for systemic toxicity (excluding reproductive and developmental toxity endpoints) were considered to be:
• 250 mg/kg/day in males based on clinical signs (hypoactivity, staggering gait, sudden startle and/or tremors) observed at 625 mg/kg/day,
• 625 mg/kg/day in females based on the absence of adverse findings before parturition.


Reproductive/developmental toxicity testing:
• In P generation animals, there was no evidence of adverse effects on mating (including precoital time), fertility, duration of gestation and number of implantation sites. However, at 625 mg/kg/day females had difficulties to deliver. These difficulties resulted in a decrease in the mean number of live pups on Day 1 p.p. associated with a decreased live birth index and an increased post-implantation loss. At 250 mg/kg/day, there was also a lower live birth index. At 250 and 625 mg/kg/day, there was an increase incidence of pups with absence of milk into the stomach and low qualitative body temperature that were considered to represent a lack of maternal care.
• In Cohort 1A and/or 1B animals there were no adverse effects on pup development (including sexual maturation) and no effects on estrous cycle or mating, but there was a decrease in female fertility index at 250 mg/kg/day which was considered to be related to the odour of the test item altering rat sexual attraction during the mating period.


There was no effect on live birth index at 250 mg/kg/day.
Therefore the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 100 mg/kg/day.


Developmental neurotoxicity testing:
• in Cohort 2A: there was no evidence of developmental neurotoxicity at any tested dose level (625 mg/kg/day was the highest dose level tested),
• in Cohort 2B: there was no evidence of developmental neurotoxicity at any tested dose level (250 mg/kg/day was the highest dose level tested).
Therefore the No Observed Adverse Effect Level (NOAEL) for developmental neurotoxicity was considered to be 250 mg/kg/day.


Developmental immunotoxicity testing:
• in Cohort 1A: there was no test item treatment-related findings on lymphocyte subtyping at any dose level (625 mg/kg/day was the highest dose level tested),
• in Cohort 3: there was no no evidence of immunotoxicity potential at any dose level (250 mg/kg/day was the highest dose level tested).
Therefore the No Observed Adverse Effect Level (NOAEL) for developmental immunotoxicity was considered to be 250 mg/kg/day.