3-(m-tert-butylphenyl)-2-methylpropionaldehyde

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

Study was enhanced by parameters which were part of the OECD Guidelines for Testing of Chemicals; No. 416 (22 Jan 2001)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF Aktiengesellschaft, Experimentelle Toxikologie und Ökologie

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-(m-tert-butylphenyl)-2-methylpropionaldehyde

EC Number:

263-580-6

EC Name:

3-(m-tert-butylphenyl)-2-methylpropionaldehyde

Cas Number:

62518-65-4

Molecular formula:

C14H20O

IUPAC Name:

3-(3-tert-butylphenyl)-2-methylpropanal

Details on test material:

- Name of test material (as cited in study report): m-Lysmeral
- Physical state: liquid/colorless, clear
- Analytical purity: > 98.4 %
- Lot/batch No.: 9336/08 S.50
- Storage condition of test material: Ambient (room temperature); under Argon
Stability of the test substance in olive oil at room temperature over a period of 7 days was investigated analytically before the beginning of the study.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: (P) x 28 (±1) days;
- Weight at study initiation: (P) Males: 127.2 - 153.3 g; Females: 95.6 - 125. 0 g
- Housing: housed individually; During overnight matings, male and female mating partners were housed together; Pregnant animals and their litters were housed together until PND 21 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: about 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

VEHICLE
- Concentration in vehicle: 1.25; 3.75; 11.25 g/100 ml
- Amount of vehicle (if gavage): 4 ml/kg body weight

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

135d (F0); 22d (F1)

Frequency of treatment:

daily

Details on study schedule:

Animals were chosen by lot and each litter was taken into account as far as technically feasible. If fewer than 25 litters were available in a group or if one sex was missing in a litter, more animals were taken from the other litters of the respective test group to obtain the required number of reared animals.
Because of offspring mortality in test group 03 (450 mg/kg body weight/day) only 10 male and 10 female F1 pups were available to be reared.
All selected F1 animals were treated with the test substance at the same dose level as their parents, from post-weaning through sexual maturity (up to about one day before they were sacrificed). The F1 rearing animals were sacrificed shortly after they reached sexual maturity.

Doses / concentrations

Remarks:

Doses / Concentrations:
0; 50; 150; 450 mg/kg bw/d
Basis:

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations:
Determined on the day of first test substance administration and then once a week. F1 rearing animals had a first collective weighing day when the last F1 litter was weaned (post-weaning day 0) and were then weighed once a week.
During gestation period the F0 generation parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 1, 4, 7, 14 and 21.
F0 parental females were not weighed during the mating interval until there was positive evidence of sperm in vaginal smears. F0 parental females without litter were not weighed during the lactation phase. Post-weaning the F0 females, waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
In general, food consumption was determined once a week for male (until 10th premating week) and female (until weaning).
During pregnancy, food consumption of the F0 females (animals with evidence of sperm) was determined weekly for GD 0 - 7, 7 - 14 and 14 - 20.
During lactation, food consumption of the F0 females with litter was determined for PND 1 - 4, 4 - 7, 7 - 14, and 14 - 21.
Food consumption was not determined during the mating period as well as for females without positive evidence of sperm and females without litter.

OTHER:
Blood samplings were carried out towards the end of the study. Blood was taken from the retro-orbital venous plexus from
fasted animals. Before sampling the animals were anaesthetized using isoflurane. Liver samples were taken at necropsy.
Hematology:
Leukocyte/Erythrocyte/Platelet/Differential blood count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin/concentration, Reticulocytes
Clinical chemistry:
Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, gamma-Glutamyltransferase, Cyaninde-insensitive Palmitoyl-CoAoxidation (in liver homogenates), Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol.

Oestrous cyclicity (parental animals):

Estrous cycle length was evaluated by daily inspection of vaginal smears for all F0 female parental rats for a minimum of 3 weeks prior to mating. The evaluations were continued throughout the pairing period until the female exhibited evidence of copulation. At the scheduled necropsy a vaginal smear was microscopically examined to determine the stage of the estrous cycle for each F0 female.

Sperm parameters (parental animals):

Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from the F0 males of all dose groups.
The following parameters were determined:
• sperm motility
• sperm morphology
• sperm head count (cauda epididymis)
• sperm head count (testis)
Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and highest dose group, only.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
Where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Standardization of litters was not performed in litters with ≤ 8 pups.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and status of delivery, pup viability/ mortality, sex ratio, sexual maturation (vaginal opening, preputial separation), pup clinical observations, pup body weight data.

GROSS EXAMINATION OF DEAD PUPS:yes
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.

Postmortem examinations (parental animals):

SACRIFICE
All F0 parental animals were sacrificed under Isoflurane anesthesia by decapitation and were exsanguinated.

GROSS NECROPSY
All F0 parental animals were necropsied and assessed by gross pathology, with particular attention on the reproductive organs.

ORGAN WEIGHTS
Liver, Kidneys, Adrenal glands, Testes, Epididymides, Cauda epididymis, Prostate, Seminal vesicles including coagulation glands, Ovaries, Uterus, Spleen, Brain, Pituitary gland, Thyroid glands (with parathyroid glands)

HISTOPATHOLOGY
Vagina, Cervix uteri, Uterus, Ovaries, Oviducts, Left testis, Left epididymis, coagulation gland, prostate, pituitary gland, adrenal glands, all gross lesions

Differential Ovarian Follicle Count:
Five sections were taken from the proximal and the distal part of both ovaries , of F0 female animals, at least 100 μm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated. Primordial follicles and growing follicles were counted by light microscope.

Postmortem examinations (offspring):

SACRIFICE
All pups with scheduled sacrifice (i.e. pups, which were culled on PND 4, and pups, which were sacrificed on PND 21 or subsequent days) were killed by means of CO2.

GROSS NECROPSY
All pups were examined externally and eviscerated; their organs were assessed macroscopically.

ORGAN WEIGTHS
Pup organ weights (brain, spleen and thymus of 1 pup/sex and litter)

Statistics:

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means
were used), estrous cycle duration, number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter, duration of sexual maturation (days to vaginal opening, days to preputial separation): DUNNETT-test (two-sided).

Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy, sexual maturation data (vaginal opening, preputial separation), males with a certain amount of abnormal sperm (cutoff value: 0.9-quantile [90%] of control groups): FISHER'S EXACT test.

Proportions of affected pups per litter with necropsy observations, Total spermatids/g testis, total sperm/g cauda epididymides, Follicles (primordial/ growing/ primordial + growing): WILCOXON-test (one- sided).

Sperm motility (%): WILCOXON-test (one- sided) with Bonferoni- Holm-Adjustment

Pup organ weights (absolute and relative), Clinical pathology parameters, Organ weight parameters: KRUSKALWALLIS- test (two-sided) + WILCOXON-test (two- sided) as post test.

Reproductive indices:

Male:
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:

Male mating Index (%) = number of males with confirmed mating*/number of males placed with females x 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility Index (%) = number of males proving their fertility*/number of males placed with females x 100
* defined by a female with implants in utero

Female:
The mating partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 females. For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:

Female mating Index (%) = number of females mated* / number of females piaced with males x 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = number of femals pregnant*/ number of females mated** x 100
* defined as the number of females with implants in utero
** deflned as the number of females with vaginal sperm or with implants in utero

Gestation Index (%) = number of females with live pups an the day of birth/ number of females pregnant* x 100
* defined as the number of females with implants in utero

The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:

Live birth index (%) = number of liveborn pups at birth / otal number of pups born x 100

The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:

Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations x 100

Offspring viability indices:

Viability and lactation indices were calculated according to the following formulas:
Viability Index (%) = number of live pups an day 4 after birth/number of live pups an the day of birth x 100
Lactation index (%) = number of live pups an day 21 after birth / number of live pups an day 4 after birth x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One female animal of the test group 450 mg/kg bw/d was sacrificed moribund on GD 16 after showing extended abdomen, salivation after treatment, piloerection and a palpable mass in left flank. Histopathology revealed a malignant mesenchymal neoplasm in the left kidney which was most likely the cause of the moribund condition of this rat. A relationship of this finding to treatment is not assumed.
All high-dose males and females and almost all mid-dose males and females (450 and 150 mg/kg bw/d) showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 15 minutes). The temporary salivation is considered test substance-induced but not an adverse toxicologically relevant finding. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract.
3 high-dose dams did not nurse their offspring properly, as no milk spots were observed in the pups.
No other test substance related effects observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
450 mg/kg bw/d:
Increased food consumption (females) during premating and gestation; decreased food consumption during whole lactation period (up to 41% below ctrl.).
Decreased body weights (males) from week 5 until the end of the study (up to 11%) and body weight gain (weeks 0 – 15, about 15%)
Decreased terminal body weights (males).
Slightly higher body weights/ gain (females) at the beginning of premating; slightly decreased weight gain during gestation and lactation with little effect on body weight.

150 mg/kg bw/d:
Decreased body weights (males) from week 10 until the end of the study (up to 6%); Decreased body weight gain (weeks 0 – 15, about 9%).
Decreased terminal body weights (males).
No other relevant adverse test substance related effects observed

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No test substance related effects observed

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No test substance related effects observed

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male fertility index ranged between 88% and 100% within the range of historical control data and without showing any relation to dosing, reflecting a normal range of biological variation inherent in the strain of rats used.
No test substance related effects observed on female mating index, mean duration until sperm, fertility index, mean duration of gestation, mean number of implantation sites.

450 mg/kg bw/d:
Decreased gestation index (86 vs. 96% in control),
Lower number of implants (10.2 vs. 12.0 in control),
Increased postimplantation loss (19.6 vs. 11.1% in control),
Decreased litter size (8.1 vs. 11.4 in control),
Increased number of dams with stillborn pups (9 vs. 0 in control),
Increased number of stillborn pups (22 vs. 0 in control),
Decreased number of liveborn pups (148 vs. 263 in control)
Decreased live birth index (87 vs. 100% in control),
2 dams with complete litter losses (all pups stillborn)

150 mg/kg bw/d:
Increased number of stillborn pups (5 vs. 0 in control),
Decreased number of liveborn pups (214 vs. 263 in control)


ORGAN WEIGHTS (PARENTAL ANIMALS)
450 mg/kg bw/d:
Increased absolute/ relative liver weights: 152%/174% males; 164%/161% females

150 mg/kg bw/d:
Increased absolute/ relative liver weights: 126%/135% males; 125%/123% females

50 mg/kg bw/d:
Increased absolute/ relative liver weights: 108%/109% males; 112%/109% females
No other relevant adverse test substance related effects observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
450 mg/kg bw/d:
Liver enlargement: 25/25 males, 21/25 females.
No other test substance related effects observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
450 mg/kg bw/d:
Liver: Hepatocellular hypertrophy in both sexes
Differential ovarian follicle count: Significant decrease of primordial, growing, and primordial growing follicles.
No other test substance related effects observed.

OTHER FINDINGS (PARENTAL ANIMALS)
HEMATOLOGY:
450 mg/kg bw/d:
Increased total white blood cell counts, absolute and relative lymphocyte counts (both sexes); increased relative reticulocyte counts (females);
Decreased relative neutrophil and eosinophil counts, red blood cell counts, hematocrit and hemoglobin in both sexes.
No other relevant adverse test substance related effects observed.

CLINICAL CHEMISTRY:
450 mg/kg bw/d:
Increased ALT, ALP, inorganic phosphate, albumin, palmitoyl CoA oxidation in liver (both sexes); Increased urea (males), triglyceride (females)
Decreased total protein, globulin, cholesterol (males); Decreased creatinine, total bilirubin (females).

150 mg/kg bw/d:
Increased ALP, palmitoyl CoA oxidation in liver (both sexes); Increased ALT (females), inorganic phosphate, urea (males)
Decreased total protein, globulin, cholesterol (males); Decreased total bilirubin (females)

50 mg/kg bw/d:
Increased ALP, palmitoyl CoA oxidation in liver (males)
Decreased total protein, globulin (males)
No other relevant adverse test substance related effects observed.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

< 50 mg/kg bw/day

Sex:

male

Basis for effect level:

other: organ weights, clinical chemistry

2

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Sex:

female

Basis for effect level:

other: organ weights, clinical chemistry

Results: F1 generation

Details on results (F1)

VIABILITY (F1 PUPS)
450 mg/kg bw/d:
Pup mortality during early lactation (viability index 55 vs. 100% in control);
Pup mortality during later lactation (lactation index 92 vs. 99% in control),
Complete litter losses in 6 F0 dams, no proper nursing in 3 F0 dams.

150 mg/kg bw/d:
Pup mortality during early lactation (viability index 96 vs. 100% in control)

VIABILITY (F1 REARING ANIMALS): No test substance related effects observed

CLINICAL SIGNS (F1 REARING ANIMALS)
All high-dose animals and 21 male and 11 female mid-dose animals (450 and 150 mg/kg bw/d) showed transient salivation during the entire treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 15 minutes). The temporary salivation is considered test substance-induced but not an adverse toxicologically relevant finding. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract.
No other test substance related effects observed.

BODY WEIGHT (F1 PUPS)
450 mg/kg bw/d:
Decreased pre-weaning pup body weights / pup body weight gain
150 mg/kg bw/d:
Decreased pre-weaning pup body weights / pup body weight gain
No other test substance related effects observed.

BODY WEIGHT (F1 REARING ANIMALS)
450 mg/kg bw/d:
Decreased post-weaning food consumption and body weights / body weight gain in males
No other test substance related effects observed.

SEXUAL MATURATION (F1 REARING ANIMALS)
450 mg/kg bw/d:
Slightly premature sexual maturity in F1 females: Earlier female sexual maturation is in line with a significantly lower body weight at commencement of vaginal patency. There was no significant adverse effect on general development in these females.
Slight delay of sexual maturation in F1 males: Later male sexual maturation corresponds to the slightly, non- significantly, lower body weights.
No effects on sex ratios observed in F1 pups.

ORGAN WEIGHTS (F1 PUPS): No relevant adverse test substance related effects observed.

GROSS PATHOLOGY (F1 PUPS/ F1 REARING ANIMALS): No relevant adverse test substance related effects observed.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

Fertility

Generation:

F1

Effect level:

450 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no adverse test substance related effects observed

2

Dose descriptor:

NOAEL

Remarks:

Developmental Toxicity

Generation:

F1

Effect level:

50 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: intrauterine/ pup mortality/ pup body weights

Overall reproductive toxicity

Reproductive effects observed:

not specified