C16-(branched), C20-(branched) and C24-(branched)-alkanes

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In the reproductive/developmental toxicity screening test in rats, the NOAEL of Tetrabutane for general toxicity as well as for reproductive/developmental toxicity was 1000 mg/kg/day (OECD TG 422, RL1, GLP; rat, oral, dose levels: 100, 300 or 1000 mg/kg bw/day; no adverse effects observed)

EOGRTS (with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)) ongoing (OECD TG 443, rat; read-across Oxooil LS9)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 December 2009 (animal arrival) -10 March 2010 (pathology completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study undertaken according to current OECD Test Guidelines.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

increased animal number

Principles of method if other than guideline:

The toxicity subgroup for this study comprised 5 male and 5 female rats per treatment group and the reproductive subgroup comprised 5 male and 10 female rats per treatment group. The 5 male rats in each treatment group of the toxicity subgroup were used for mating with 5 female reproductive subgroup animals of the corresponding treatment group. The ten female rats in each treatment group of the reproductive subgroup were used exclusively for the reproductive/developmental toxicity phase of the study. This deviation was undertaken to enhance the robustness of the study.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 303-355 g
- Fasting period before study: none
- Housing: Male: 5 animals/P2000 polycarbonate cage : singly with one female in RB3 modified polyproplyene cages during mating.
Females: 5 animals/P2000 polycarbonate cage pre-mating: singly with one male in RB3 modified polypropylene cages during mating: singly in 2154 polycarbonate cages during gestation and littering
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 2 December 2009 (animal arrival) To: 20 January 2010 (necropsy completion)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was used as supplied. All formulations were prepared freshly weekly and were stored refrigerated (2-8°C) in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 20, 60 or 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): no data
- Purity: no data

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until detection of mating (successful for all animals up to 5 days)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): singly in 2154 polycarbonate cages
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix. The stability was assessed following storage at ambient temperature (nominally 21°C) for 0 hours, 4 hours (continual stirring) and 2 days, and refrigeration (nominally 2-8°C) for 2, 8 and 15 days. Prior to initial sampling on each day, the formulation was mixed by 20-fold inversion and magnetic stirring for a minimum of 5 minutes. At each time point, single samples (nominally 1 mL) were taken for assay from the top, middle and bottom of the magnetically stirred formulation. Stability was determined from the mean concentration of the analyte in the vehicle at each sampling point. Specimen formulations (typically 400 mL) were prepared at concentrations of 2 and 200 mg/mL and equally split between four amber glass screw-capped bottles and were confirmed for 15 days when refrigerated and for 48 hours at room temperature.
Samples of each formulation prepared for administration in the first week of the dosing procedure were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed); 2 assays from each group and 1 assay. The remainder was frozen (nominally -20°C) and retained as contingency for analysis if any result required confirmation.

The GC analytical procedure was validated with respect to linearity of detector response, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision.

The homogeneity and stability was confirmed for Tetrabutane in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for 2 days and refrigerated storage for up to 15 days. The storage times represented the maximum time from preparation to completion of administration.

The mean concentrations of Tetrabutane in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation

Duration of treatment / exposure:

5 weeks or for dams up to day 4 of lactation (daily)

Frequency of treatment:

All animals were dosed once each day at approximately the same time each day, seven days per week.

Details on study schedule:

Age at mating of the mated animals in the study:11-12 weeks

Remarks:

Doses / Concentrations:
100, 300 or 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 10 females in the reproductive toxicity subgroup, and 5 males used for mating from the toxicity subgroup

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels of 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor with reference to previous work with this compound performed in these laboratories (Huntingdon Life Sciences Report Number: BBB0024). In that study, the CD rats received Tetrabutane at doses of 100, 300 or 1000 mg/kg/day for seven days, there were no findings which precluded the use of these dose levels on the subsequent 4 week general toxicity and reproductive developmental toxicity screening study.

- Rationale for animal assignment (if not random):
On arrival, the animals were removed from the transit boxes and allocated to study cages. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately
- Rationale for selecting satellite groups: no satellite groups used
- Post-exposure recovery period in satellite groups: no post-exposure recovery period

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

- Time schedule:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Immediately before dosing
Immediately after dosing on return of the animal to its cage
On completion of dosing of each group
Between one and two hours after completion of dosing of all groups
As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Before treatment commenced , during each week of treatment and for females on Days 0, 7, 14 and 20 after mating and Day 4 of lacation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware (as far as practically possible) of the experimental group to which the animal belonged. For females after mating, a small number of animals were subject to these observations on each Day 4 of lactation (8, 15, 4, 8 and 4 females on each Day 4 of lactation) - the observer was aware of the experimental group and this was considered acceptable.

After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.


BODY WEIGHT: Yes

- Time schedule for examinations:
All toxicity and reproductive subgroup males (10 animals per treatment group) were weighed weekly throughout the study. All toxicity and reproductive subgroup females (15 animals per treatment group) were weighed weekly for the first two weeks and the toxicity subgroup females (five animals per treatment group) were weighed during Weeks 3, 4 and 5.

FOOD CONSUMPTION: Yes

The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded during weeks 1 and 2 for all male (two cages of 5 animals per treatment group) and all female (three cages of 5 animals per treatment group) toxicity and reproductive subgroups. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

WATER CONSUMPTION : Yes

Fluid intake was assessed by daily visual observation. No effect was observed and, consequently, quantitative measurements were not performed

Oestrous cyclicity (parental animals):

For two weeks before pairing, daily vaginal smears were taken from females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed

Sperm parameters (parental animals):

Not undertaken

Litter observations:

All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter. The records maintained were as follows:

Clinical signs: Daily records were maintained for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.

Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-4 of age.

Sex ratio: The sex ratio of each litter was recorded on Days 1 and 4 of age.

Bodyweight: Individual offspring bodyweights were recorded on Days 1 and 4 of age.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all animals sacrificed after 5 weeks of treatment
- Maternal animals: all surviving animals sacrificed on Day 4 of lactation. One dam and litter were killed on Day 2 of lactation for reasons of animal welfare

GROSS NECROPSY

All animals were subject to a detailed necropsy. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative


ORGAN WEIGHTS

The following organs, taken from all animals, were dissected free of adjacent fat and other contiguous tissue and the weights recorded:

Epididymides (L&R) Seminal vesicles and coagulation gland
Ovaries with oviducts (L&R) Testes (L&R)
Prostate Uterus with cervix

L&R Bilateral organs weighed individually

Organ weights were also adjusted for terminal bodyweight using the weight recorded before necropsy

HISTOPATHOLOGY

The following tissues were examined for all animals of the Control and 1000 mg/kg/day treatment groups sacrificed on completion of the scheduled treatment period:

Epididymides (L&R) Seminal vesicles and coagulation gland
Mammary area - caudal† Testes (L&R)
Ovaries with oviducts Uterus with cervix
Prostate Vagina


For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted. For the assessment of the ovaries, one mid-line section of each ovary was examined for the presence of primordial follicles, growing follicles and corpora lutea

Postmortem examinations (offspring):

SACRIFICE
- F1 offspring were killed on Day 4 of age

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY

For F1 offspring surviving to scheduled termination, a full macroscopic examination of the tissues was performed, after a review of the history. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

For premature adult deaths before weaning, missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before weaning were examined as detailed above except that the cranium was not sectioned unless required to investigate a cranial abnormality. The necropsy also included an assessment for the presence of milk in the stomach, where this was possible.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not undertaken

Statistics:

See 'materials and methods' free text field below

Reproductive indices:

Mating performance and fertility
Gestation length

Offspring viability indices:

Survival indices
Sex ratio

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs, arena observations or signs related to dosing considered to be related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female control animal was killed for welfare reasons on Day 2 of lactation after signs of piloerection, pallor, hunched posture, deep breathing and reduced body temperature. Macroscopic examination at necropsy revealed yellow amorphous adhesions on the lungs and bronchi, enlarged thymic lymph nodes, clear fluid in the thorax, and brown staining of the fur in the uro-genital region. Microscopic examination revealed evidence of oesophageal perforation, thereby indicating that the death of this Control female was due to a dosing error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The bodyweight and bodyweight change of females receiving 1000 mg/kg/day was slightly high during Days 0-7 and 14-20 of gestation when compared with that of the Control, resulting in a slightly higher overall bodyweight gain during gestation for females receiving 1000 mg/kg/day. Bodyweight change from Day 1 to Day 4 of lactation was similar across all groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no findings that were considered to be related to treatment with Tetrabutane

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no findings that were considered to be related to treatment with Tetrabutane

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There was a higher incidence of acyclic animals amongst females treated with Tetrabutane at 100 or 300 mg/kg/day (4/10 and 3/10 at 100 and 300 mg/kg/day, respectively, compared with 1/10 in the concurrent Control group). However, as all females mated within 5 days, and there no acyclic females in the 1000 mg/kg/day group, oestrous cycles were considered unaffected by treatment.

The majority of animals mated within 4 days (i.e. at the first oestrus opportunity after pairing). Two females mated within 5 days.

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

Not undertaken

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance and fertility were unaffected by treatment.
The duration of gestation was unaffected by treatment, with all females having a gestation length within the expected range of 22-23 days. The gestation index was 100% in all groups

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive/developmental toxicity up to a limit dosage of 1000 mg/kg/day

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs amongst the offspring which were considered related to maternal treatment with Tetrabutane

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of uterine implantation sites, total litter size at Day 1 of age and live litter size at Days 1 and 4 were unaffected by parental treatment.
Neither peri- nor post-natal offspring survival was affected by parental treatment with Tetrabutane

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Assessment of offspring bodyweight data showed that bodyweight at Day 1 of age and subsequent growth to Day 4 appeared slightly low for the offspring of all treated groups. This is likely to be a consequence of the slightly higher litter size in these groups, and not selectively related to the parental administration of Tetrabutane.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of the small number of offspring dying before scheduled termination on Day 4 of age showed absence of milk in the stomach as the predominant finding. This is a common observation for offspring dying at such a young age and was considered not to indicate any adverse effect of maternal treatment with Tetrabutane.
There were no treatment related findings among offspring examined at scheduled termination on Day 4 of age.

Histopathological findings:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive/developmental toxicity up to day 4 of lactation

Key result

Reproductive effects observed:

no

TABLE 1

 

Bodyweight change – group mean values (g) for reproductive subgroup females during gestation and lactation

 

Group	:	1	2	3	4
Compound	:	Control	Tetrabutane	Tetrabutane	Tetrabutane
Dose (mg/kg/day)	:	0	100	300	1000

 

			Gestation					Lactation
Group			Days	Days	Days	Days		Days
/Sex			0-7	7-14	14-20	0-20		1-4
Statistical test:			Wi	Wi	Wi	Wi		Wi
1F	Mean		35	34	77	145		9
	SD		3.7	6.3	14.5	15.6		4.3
	N		10	10	10	10		9
2F	Mean		32	33	80	145		6
	SD		6.8	6.4	10.2	14.8		6.3
	N		10	10	10	10		10
3F	Mean		31	32	84	147		11
	SD		5.6	6.1	6.9	12.0		10.2
	N		10	10	10	10		10
4F	Mean		41	35	87	162*		9
	SD		9.7	7.8	13.3	24.1		6.6
	N		10	10	10	10		10

 

Wi    Treated groups compared to Control using Williams’ test.

 

 

Conclusions:

Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive/developmental toxicity within the scope of this screening test was 1000 mg/kg/day.

Executive summary:

An OECD 422 guideline screening study was performed at the Laboratories of Huntingdon Life Sciences, Eye, on behalf of Evonik Oxeno GmbH., to investigate the reproductive and developmental toxicity of the test substance Tetrabutane when administered to rats by oral gavage. Three groups, each comprising five male and ten female rats, received Tetrabutane once daily at dosages of 100, 300 or 1000 mg/kg/day. An additional five males from each treatment group of the toxicity phase of this combined study were used for mating with corresponding females of the reproductive phase. The males were treated daily for a period of five complete weeks before termination. The females were treated daily for two weeks before pairing, throughout pairing and gestation and for three days of lactation, until termination on Day 4 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose, for the same duration.

Throughout the study, data was recorded on clinical condition, detailed physical and arena observations and bodyweight. Data was recorded on food consumption for all animals prior to mating, and during gestation and lactation for mated females. For females, data were recorded on oestrous cycles, mating performance and fertility and gestation length.  Organ weight, macroscopic and microscopic pathology investigations were undertaken. The clinical condition of F1 offspring, litter size and survival, sex ratio and bodyweight were assessed and macroscopic pathology investigations were undertaken. The study was conducted to GLP.

There was no effect of treatment on mating performance, fertility or gestation and there were no pathology findings or changes considered to be related to treatment. There was a slightly increased overall bodyweight gain during gestation for females receiving 1000 mg/kg/day, when compared with controls. Bodyweight change from Day 1 to Day 4 of lactation was similar across all groups.

The reproduction/developmental toxicity screening test found no evidence of impaired performance at 100, 300 or 1000 mg/kg/day Tetrabutane. Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive/developmental toxicity within the scope of this screening test was 1000 mg/kg/day.

The study is considered acceptable for classification and satisfies the guideline requirements for a rat reproductive/developmental toxicity screening test.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A Reproduction / Developmental Toxicity Screening Test was conducted with the target substance Tetrabutane. Screening tests are also available for the source substances Oxooil LS9 and Oxooil LS13. An Extended One Generation Reproduction Toxicity Study is currently ongoing with the source substance Oxooil LS9. A justification for read-across is attached to Iuclid section 13.

 

Study with the target substance

An OECD 422 guideline screening study was performed to investigate the reproductive and developmental toxicity of the test substance Tetrabutane when administered to rats by oral gavage. Three groups, each comprising five male and ten female rats, received Tetrabutane once daily at dosages of 100, 300 or 1000 mg/kg bw/day. An additional five males from each treatment group of the toxicity phase of this combined study were used for mating with corresponding females of the reproductive phase. The males were treated daily for a period of five complete weeks before termination. The females were treated daily for two weeks before pairing, throughout pairing and gestation and for three days of lactation, until termination on Day 4 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose, for the same duration.

Throughout the study, data was recorded on clinical condition, detailed physical and arena observations and bodyweight. Data was recorded on food consumption for all animals prior to mating, and during gestation and lactation for mated females. For females, data were recorded on oestrous cycles, mating performance and fertility and gestation length.  Organ weight, macroscopic and microscopic pathology investigations were undertaken. The clinical condition of F1 offspring, litter size and survival, sex ratio and bodyweight were assessed, and macroscopic pathology investigations were undertaken.

There was no effect of treatment on mating performance, fertility or gestation and there were no pathology findings or changes considered to be related to treatment. There was a slightly increased overall bodyweight gain during gestation for females receiving 1000 mg/kg/day, when compared with controls. Bodyweight change from Day 1 to Day 4 of lactation was similar across all groups.

The reproduction/developmental toxicity screening test found no evidence of impaired performance at 100, 300 or 1000 mg/kg/day Tetrabutane. Based on the results of this study, it was concluded that the NOAEL for reproductive/developmental toxicity within the scope of this screening test was 1000 mg/kg bw/day.

 

Studies with the source substances

A Reproduction / Developmental Toxicity Screening Test in accordance with OECD Guideline 421 (2016) was conducted with the source substance Oxooil LS13 at dose levels of 0, 150, 500 and 1000 mg/kg bw/d.

In this study, salivation was the most relevant clinical sign recorded for males and females, observed in all treated groups with a dose-related incidence.

No relevant toxicological changes were observed in thyroid hormones in treated males, when compared to controls. Changes observed in thyroid hormones in pups (day 14 post partum) of treated dams were considered to be unrelated to treatment.

Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show relevant intergroup differences. Implantation sites, pre-implantation loss data, pre-natal loss data and gestation length of females Implantation, pre-implantation and pre-natal data were similar between treated and control groups, with the exception of a statistically significantly higher pre-natal loss observed in females dosed at 1000 mg/kg/day, compared to controls. All pregnant dams gave birth on Day 22 post coitum (mean value).

Reductions in total and live litter size (up to -20%) and in mean litter weight (-26%), the latter statistically significant on Day 4 post partum, were seen in females receiving 1000 mg/kg/day, compared to controls, at birth and on Days 1 and 4 post partum.

In addition, on Day 13 post partum, slight, but statistically significant decreases in mean litter and mean pup weights (up to -16%) were seen in high dose females, compared to controls.

No relevant intergroup differences were seen in the sex ratio in treated groups compared to controls.

Pre-weaning clinical signs were comparable between treated and control groups. An increase in the number of pups found dead in cage, the majority on Days 0-1post partum, was seen in litters of treated females compared with controls.

No toxicological significance was attributed to the slight, without dose-relationship and within the historical control data, statistically significant increases seen in anogenital distance in male pups from treated females, when compared to controls; no relevant differences were observed in female pups.

No nipples were seen in pups on Day 13 post partum.

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect. No nipples were observed in male pups on Day 14 post partum.

No relevant differences in thyroid weight were seen in male pups from treated females compared to controls, while slight differences were observed in mean thyroid weight of female pups from mid and high dose females (-11% and -17%, respectively).

No changes were observed on terminal body of study animals of both sexes, when compared to the control group. A statistically significant increase was observed in mean liver weight of mid and high dose animals of both sexes, in mean kidney weight of mid and high dose males and in mean adrenals weight of mid and high dose females.

The only relevant change observed following gross pathology examination was enlarged kidneys of two mid dose males.

Treatment-related changes, associated with the oral administration of the test item, were present in the non-glandular region of the stomach of mid and high dose animals of both sexes, in liver and kidneys of mid and high dose males, in liver of high dose females. As consequence of the above mentioned treatment-related findings, the cortical hypertrophy of the adrenals of females receiving 500 and 1000 mg/kg/day could be considered stress-related. No other treatment-related changes were noted in animals sacrificed at the end of the treatment period. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 150 mg/kg/day for males and females, as at this level no histopathological changes were observed; while the NOAEL for reproductive and developmental toxicity was considered to be 500 mg/kg/day, since at level of 1000 mg/kg/day were seen effects in litter data in presence of maternal toxicity (body weight changes and histopathological findings).

 

A Reproduction / Developmental Toxicity Screening Test (OECD TG 422) was also performed with the source substance Oxooil LS9 with dose levels of 0, 100, 300 or 1000 mg/kg/day for a period of five complete weeks before termination. An additional five male and ten female rats per treatment group were used to investigate the reproductive toxicity potential of Oxooil LS9. The males were treated daily for a period of five complete weeks before termination. The females were treated daily for two weeks before pairing, throughout pairing and gestation and for three days of lactation, until termination on Day 4 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose, for the same duration.

Throughout the study, data were recorded on clinical condition, detailed physical and arena observations and bodyweight. Data were recorded on food consumption for all animals prior to mating, and during gestation and lactation for mated females. For females, data were recorded on oestrous cycles, mating performance and fertility and gestation length.  Organ weight and macroscopic and microscopic pathology investigations were undertaken. The clinical condition of F1 offspring, litter size and survival, sex ratio and bodyweight were assessed and macroscopic pathology investigations were undertaken.

Three of ten males treated at 1000 mg/kg/day were sacrificed on humane grounds during weeks 3 or 4 and the remaining males in this group killed prematurely in week 4 (Section 7.5.1). Two females treated at this dosage were killed for welfare reasons on Day 20 of gestation or Day 3 of lactation, respectively. Bodyweight loss and reduced food consumption were recorded for males receiving 1000 mg/kg/day and reduced bodyweight gain for males treated at 300 mg/kg/day and dams receiving 1000 mg/kg/day.

There was no effect of treatment on mating performance, fertility, reproductive performance, survival or development of the offspring, and there were no treatment-related pathological findings in the reproductive organs. Slightly lower male and female offspring mean bodyweights were recorded for the litters of females that received 1000 mg/kg/day, which were considered to be a consequence of the low terminal bodyweight of females that received 1000 mg/kg/day and indicative of the toxicity in the dams at this dosage.

Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) in rats for reproductive toxicity within the scope of this screening test is 1000 mg/kg/day.

 

Overall, no toxicity to reproduction in the absence of maternal toxic effects was observed in the available studies conducted with the target and the source substances.

 

 

Extended One-Generation Reproduction Toxicity Study

An extended one generation reproduction toxicity study with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B) in rat is currently ongoing with the source substance Oxooil LS9. When the results are available, the dossier will be updated accordingly. Based on the assessment of toxicological data for the source and target substance a read across for extended one generation toxicity study is considered reliable as laid down in the read across justification attached to Iuclid section 13.

Effects on developmental toxicity

Description of key information

In the prenatal developmental toxicity study in rat, the NOAEL was 300 mg/kg b.w./day for the dams. The NOAEL for the fetal organism was 300 mg/kg b.w./day (read-across from Oxooil LS9).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and target substance have similar toxicological properties because they
- are manufactured from the same starting material in a similar process
- have similar chemical structures with common functional groups: the target and source substances are mainly branched hydrocarbons, with however, differences in C-chain length.
- have similar physico-chemical properties,
- have the same mode of action, which is well-established for low molecular weight hydrocarbons

This read-across hypothesis corresponds to scenario 2 - different compounds have qualitatively and quantitatively similar properties - of the read-across assessment framework i.e. properties of the target substance Tetrabutane are predicted to be similar to those of the source substance Oxooil LS9.

Based on the available experimental data, the read-across strategy is supported by close structural analogy as well as similar toxicological data.

Therefore, read-across from the existing reproduction toxicity and developmental toxicity studies conducted with the source substance Oxooil LS9 is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance Tetrabutane, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see Justification for Read-Across attached to Iuclid section 13

3. ANALOGUE APPROACH JUSTIFICATION
see Justification for Read-Across attached to Iuclid section 13

4. DATA MATRIX
see Justification for Read-Across attached to Iuclid section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Species:

rat

Strain:

other: CD® / Crl:CD(SD)

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Treatment period: Day 6 to 20 of gestation

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Dose / conc.:

100 mg/kg bw/day

Remarks:

low dose

Dose / conc.:

300 mg/kg bw/day

Remarks:

intermediate dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

high dose

Control animals:

yes, concurrent vehicle

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At the high dose level (1000 mg Oxooil LS9/kg b.w./day) several dams were noted with signs of clinical toxicity on several test days (e.g. a reduced motility (17 of 22), salivation (21 of 22), prone position (3 of 22), piloerection (3 of 22), pale faeces (9 of 22) and an increased (4 of 22) or decreased water consumption (2 of 22)).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the high dose level (1000 mg Oxooil LS9)/kg b.w./day) a statistically significantly reduced body weight was noted from gestation day 10 until laparotomy on gestation day 21 (max.: 15.0% below the value of the control group at the day of laparotomy). Body weight gain for the whole study period was 49.7% for the dams of the high dose group in comparison to 78.4% for the dams of the control group (p ≤ 0.01).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant (p ≤ 0.01) reductions in food consumption were noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day) on several gestation days after the start of treatment (max. 36.4% below the value of the control group between gestation days 6 and 7).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

At the high dose level (1000 mg Oxooil/kg b.w./day) 4 dams with an increased water consumption and 2 other dams with a decreased water consumption were noted by visual appraisal.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Uterus and carcass weights: At the high dose level (1000 mg Oxooil LS9/kg b.w./day) statistically significant reductions were noted for the gravid uterus weight (22.4% below the value of the control group) and the carcass weight (12.5% below the value of the control group).

Further organ weights: The weighing of the heart, the kidneys and the liver revealed no test item-related differences between the dams of the control group and the treatment groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

A total resorption of all implants (a postimplantation loss of 100%) was noted for 2 of 22 dams of the high dose group (1000 mg Oxooil LS9/kg b.w./day), leading to a statistically significantly (p ≤ 0.01) increased post-implantation loss (10.9% in comparison to 4.7% in the control group).

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

pre and post implantation loss

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly (p ≤ 0.01) decreased fetal weight was noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day). The fetal weight 13.4% below the value of the control group (male and female fetuses together).

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

not examined

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No malformation was noted during the macroscopic examination at laparotomy (external inspection and inspection of the inner organs and tissues for gross lesions).

Skeletal malformations:

no effects observed

Description (incidence and severity):

No malformation was noted during the skeletal examination according to DAWSON.

Visceral malformations:

no effects observed

Description (incidence and severity):

No malformation was noted during the soft tissue examination according to WILSON.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly (p ≤ 0.01) decreased placental weight was noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day). The placental weight at the high dose level was 9.9% below the value of the control group (male and female placentae combined).

Retardations: At the high dose level (1000 mg Oxooil LS9/kg b.w./day) the skeletal examination according to DAWSON revealed an increased incidence of delays in ossification for the metacarpalia and the sternebra(e) that are considered to be test item related.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: retardations: Increased incidence of delayed ossifications of the metacarpalia and the sternebra(e) at the high dose level

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg/kg b.w./day for the dams.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was 300 mg/kg b.w./day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No prenatal developmental toxicity study in rat or rabbit is available for the target substance Tetrabutane, however, a study in rat was conducted with the source substance Oxooil LS9. In addition, the conduct of a prenatal developmental toxicity study in rabbit is proposed with the source substance Oxooil LS9. The dossier will be updated after the report is available. A justification for read-across is attached to Iuclid section 13.

 

Studies with the target substance

No data available

 

Studies with the source substances

In a prenatal developmental toxicity study, the test item Oxooil LS9 was administered orally to female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg Oxooil LS9 /kg b.w./day for the dams.

None of the dams died prematurely. Noteworthy clinical observations were noted only at the high dose level (1000 mg /kg b.w./day) in form of e.g. reduced motility, prone position and piloerection.

In addition, a reduced body weight and reductions in food consumption were noted at the high dose level (1000 mg /kg b.w./day).

No test item-related changes were noted during the macroscopic inspection at necropsy and for the organ weights of the heart, the kidneys and the liver.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was 300 mg/kg b.w./d.

At the maternal toxic dose level of 1000 mg/kg b.w./day 2 dams were noted with a total implantation loss. Furthermore, reduced placental and fetal body weights as well as an increased incidence of delayed ossifications of the metacarpalia and the sternebra(e) were noted at the high dose level. These dose-related findings are considered to be treatment-related. No dead fetuses, no malformations and no test item-related increase in the incidence of variations were noted during the examination of the fetuses.

 

Based on the read-across hypothesis, these results obtained with the source substance Oxooil LS9 are also applicable to the target substance Tetrabutane.

A prenatal developmental toxicity study in rabbit was proposed for the source substance Oxooil LS9. When the results are available, the dossier will be updated accordingly. Based on the assessment of toxicological data for the source and target substance a read across for the prenatal developmental toxicity study in rabbit is considered reliable as laid down in the read across justification attached to Iuclid section 13.

Justification for classification or non-classification

No classification for reproductive toxicity is indicated according to the general classification and labelling requirements for dangerous substances and preparations (Directive 67-548-EEC) or the classification, labelling and packaging (CLP) regulation (EC) No 1272/2008.