tert-butyl acrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A combined sub-chronic toxicity study with a reproduction / developmental toxicity screening test (OECD422) with tert-butyl acrylate did not reveal any potential of the substance to impair fertility. This is supported by the results of a two-generation study with the structural analogue methyl acrylate and subchronic, chronic studies and an extended one generation study with n-butyl acrylate.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Aug 2002 - 04 Dec 2003 (experimental)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 413 (Sub-Chronic Inhalation Toxicity: 90-day Study)

Version / remarks:

May 12, 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 22, 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental)

Version / remarks:

712-C-00-368; July, 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

712-C-98-204; August, 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC L133 (Sub-Chronic Inhalation Toxicity Study)

Version / remarks:

May 30, 1988

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Tert.- Butylacrylat
- Physical state: colorless liquid
- Analytical purity: 99.62% (Batch No. 2-011002-15/1) - 99.8% (Batch No. B602)
- Purity test report: Analytical report 02L00206 (Batch No. B602); reanalysis (Batch No. 2-011002-15/1)
- Stability under test conditions: The stability under storage conditions over the exposure period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: refrigerator, protected from light and stored under air

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: ca. 4 weeks
- Weight at study initiation: Males: 106.1-108.3 g; Females: 92.7-94.7 g (groupwise)
- Housing: individually in type DK II stainless steel wire mesh cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Underneath the DK III cages, waste trays were fixed containing bedding material (type 3/4 dust free embedding (SSNIFF, Soest, FRG); from day 18 post coitum until sacrifice, the pregnant animals and their litters were housed in Markolon M III cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Pregnant females were provided with nesting material (cellulose wadding) towards the end of pregnancy.
- Diet: ad libitum, "GLP" (Provimi Kliba SA, Kaiseraugst, CH)
- Water: ad libitum, tap water
- Acclimation period: ca. 2 weeks

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

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: clean air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Piston metering pumps (Sarstedt DESAGA) and glass vaporizers with thermostat (BASF AG)
- Method of holding animals in test chamber: The animals were kept singly in wire cages located in a glass-steel inhalation chamber, volume of 1.4 m3 (BASF AG).
- Method of conditioning air: For each concentration the test substance was supplied to a thermostated vaporizer at a constant rate by means of the metering pump. The vapor was generated with conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C) and passed into the inhalation system.
- Temperature, pressure in air chamber: 25 ± 3°C, -10 Pa
- Air flow rate: 27.5-28.5 m3/h

TEST ATMOSPHERE
- Brief description of analytical method used:  The concentrations of the inhalation atmospheres were analyzed by gas chromatography in all test groups including control (Hewlett-Packard 5840 A). Daily means were calculated based on 2 measured samples per concentration and exposure. From the daily mean values of each concentration, mean  concentrations and standard deviations for the entire study were derived. The concentration constancy in each inhalation system was continuously monitored by means of a total hydrocarbon analyzer.
To ensure, that no liquid aerosols were formed at concentration levels as high as 180 ppm, a scattered light photometer was used to monitor the test atmosphere of the high dose group.
- Samples taken from breathing zone: yes

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: from study day 69 - study day 80
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Target concentrations were: 0.106, 0.319, and 0.956 mg/L (corresponding to 20, 60, and 180 ppm)
- Measured concentrations were: 0.107 ± 0.0061, 0.317 ± 0.0211, 0.958 ± 0.0481 mg/L

Duration of treatment / exposure:

- males: ca. 13 weeks (10 weeks premating, 3 weeks mating and post mating)
- females: ca. 15 weeks (10 weeks premating, during mating and gestation through day 4 after delivery)

Frequency of treatment:

6 hours/day, 5 days/week

Details on study schedule:

- Age at mating of the mated animals in the study: 16 weeks
- After ten weeks of exposure, the parental animals were mated to produce a litter. Mating pairs were formed from the same concentration group. The parental animals were examined for their mating and reproductive performances.
- The pups were sexed and were weighed on the day after birth and on day 4 post partum. Their viability was recorded. All pups were necropsied on day 4 post partum and were examined macroscopically for external and visceral findings.

Dose / conc.:

0.106 mg/L air (nominal)

Remarks:

equals 20 ppm

Dose / conc.:

0.319 mg/L air (nominal)

Remarks:

equals 60 ppm

Dose / conc.:

0.956 mg/L air (nominal)

Remarks:

equals 180 ppm

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
0.106 mg/L (ca. 20 ppm) : as the expected no observed adverse effect level
0.319 mg/L (ca. 60 ppm) : as the intermediate dose level
0.956 mg/L (ca. 180 ppm): as the dose level where toxic effects were expected

Preflow period of 4 days

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at least 3 times (before, during and after exposure) on exposure days and once during the preflow period, on the day of neurofunctional test and prior to gross necropsy. During exposure only a group wise examination was possible. The nesting, littering, and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. The littering behavior of the dams was also inspected on each workday in the afternoons in addition to the evaluations in the mornings.

BODY WEIGHT:
- Time schedule for examinations: day -7, on day -4 (start preflow period), on day 0(start exposure period) and then in weekly intervals as well as prior to gross necropsy.

FOOD CONSUMPTION:
- Time schedule: day -7, on day -4 (start preflow period), on day 0 (start of exposure period) and then in weekly intervals.
- It was calculated as mean food consumption in grams per animal and day.
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

HAEMATOLOGY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular, haemoglobin concentration, platelets, differential blood count, prothrombin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium, bile acids

URINALYSIS:
- Time schedule for collection of urine: not reported
- Analysis only performed in males
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined: volume, colour, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment

NEUROBEHAVIOURAL EXAMINATION:
- Detailed clinical observation (DCO) were performed in all animals prior to the exposure period and thereafter in weekly intervals. The findings were ranked according to the degree of severity, if applicable. The following parameters were examined: abnormal behaviour during handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, faeces (appearance/consistency), urine, pupil size
- A functional observational battery (FOB) was carried out on the assigned animals (5 males and 5 females/ test group) on study days 56 and 57 for males and females, respectively. On the days of neurofunctional tests there was no exposure of the concerning animals as well as the other 5 animals of the same test group. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. During the home cage observations attention was paid to posture, tremor, convulsions, abnormal movements, impairments of gait and other findings. During the open field observations the following parameters were examined: behaviour when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, faeces (number of faecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within 2 minutes and other findings. After the open field test animals were subjected to the following sensorimotor or reflex tests: approach response, touch response, vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behaviour during “handling”, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test and other finding. All findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Motor activity (MA) was measured on the same day and with the same animals as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order . The measurements started at about 14:00 p.m. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages ; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

Oestrous cyclicity (parental animals):

not reported

Sperm parameters (parental animals):

not reported

Litter observations:

PARAMETERS EXAMINED:
The following parameters were examined in F1 offspring: number and sex of pups, weight gain (The pups were weighed on the day after birth (day 1 p.p.) and on day 4 after birth), stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals 3 weeks after mating
- Maternal animals: All surviving animals after d4 of gestation

GROSS PATHOLOGY:
The animals were sacrificed under narcoren® anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology. To prevent post mortem autolysis, the animals that died intercurrently were necropsied as soon as possible after death.

ORGAN WEIGHTS:
The following weights parameters from all animals sacrificed were determined: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, uterus, thymus, spleen, brain, heart, lungs

HISTOPATHOLOGY:
-The following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, spinal cord (cervical, thoracic and lumber cord), sciatic nerve, pituitary gland, salivary glands (glandula mandibularis and glandula sublingualis), thyroid glands/parathyroid glands, adrenal glands, prostate gland, seminal vesicles, coagulation glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes (mandibular and mesenteric), spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, oesophagus, stomach (forestomach and glandular stomach), duodenum, jejunum ileum, caecum, colon, rectum, urinary bladder, sternum with marrow, bone marrow (femur), skull (with nasal cavities, larynx, pharynx, eyes with optic nerve, femur with knee joint, skin, skeletal muscle, extraorbital lacrimal glands. Testes, epididymides and ovaries of animals that were killed as scheduled were fixed in Bouin's solution and embedded in paraplast, thereafter . Testes, epididymides and ovaries of animals that died intercurrently were fixed in 4% formaldehyde solution.
- After the organs were fixed, histotechinical processing and examination was were performed as follows: Nasal cavities (level I- IV), Larynx (level I- III), Trachea (longitudinal, with carina), Lungs (5 lobes) and thyroid glands/parathyroid glands in all animals; all gross lesions in all affected animals; evaluations of all other organs and tissues fixed were only performed in animals of the control and high dose group

Postmortem examinations (offspring):

GROSS NECROPSY
All surviving pups (after sacrifice on day 4 p .p . by means of C02), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

Two-sided Dunnett test for food consumption, body weight and body weight change, number of mating days, duration of gestation, number of pups
delivered per litter.
Pairwise comparison by the Fisher´s exact test for male and female mating index, male and female fertility index, 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 and urine analysis except vlume, color, turbidity and specific gravity.
Pairwise comparison by the Wilcoxon test for the proportions of affected pups per litter with necropsy observations.
Non-parametric Kruskal-Wallis test (two-sided)/Wilcoxon test for feces, rearing, grip strength forelimbs, grip strength hindlimbs, landing foot-splay test, motor activity for the different time intervals, clinical pathology parameters except differential blood count and organ weights.

Reproductive indices:

- Male mating index (%) = number of males with confirmed mating / number of males places with females x 100
- Male fertility index (%) = number of males proving their fertility / number of males place with females x 100
- Female mating index (%) = number of females mated / number of females placed with males x 100
- Female fertility index (%) = number of females pregnant / number of females mated x 100
- Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x 100

Offspring viability indices:

- Viability index (%) = number of live pups on day 4 after birth / number of liveborn pups on the day of birth x 100
- Sex ratio (%) – number of live male or female pups on day (0/4) / number of live male and female pups on day (0/4) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the study period one male animal of the control group showed injuries laterally at right flank and on the right head. This injury was of mechanic matter and was therefore not related to the study. One female animal of the low concentration group showed alopecia on dorsal body region and on both forelimbs. This was most likely to be incidental, because alopecia was not observed in other animals of the low and mid concentration group. At the high concentration (0.956 mg/L) male and female animals showed various clinical abnormalities comprising slight to moderate visually increased respiration, eyelid closure, salivation, eye discharge (red) indicating that the test substance was irritating to eyes and upper respiratory tract at this high concentration. Other findings like aggressiveness, apathy (1 female), as well as alopecia and piloerection were more of general nature indicating the bad general state of the animals.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm):
Unspecific clinical symptoms indicative for some irritation and systemic toxicity (comprising visually increased respiration, salivation, piloerection, eyelid closure, eye discharge, alopecia, aggressiveness, hyperactivity and apathy)

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Two female animals exposed to the high concentration died on study day 88 and 91 (day 18 and 20 of gestation), respectively. Both animals were found pregnant at death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development of the high concentration animals was substantially impaired by the exposure to the test substance. Although the mean body weights on some examination days were not of statistical significance, the significantly reduced mean body weight gains proved the existence of this effect. The retardation of the body weight development is not only of secondary matter due to the reduced food consumption.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm):
Test group 3 (0.956 mg/l = 180 ppm):
Retarded body weight development of the males
- mean body weight: - 8.1 % to - 15.5 % of the control from study day 9 onward (statistically significant to a level of 99 %)
- mean body weight gain: -23.2 % to - 36.3 % of the control from study day 9 onward (statistically significant to a level of 99 %)
Retarded body weight development of the females
- mean body weight: - 3.0 % to -8.0 % of the control from study day 9 onward (statistically significant on study day 51 to a level of 95 %)
- mean body weight gain: -19.9 % to - 41.8 % of the control from study day 9 onward (statistically significant to a level of 99 %)
Significantly (to a level of 99 %) decreased mean terminal body weight in males and in females (- 17.2 % and -9.4 %, resp.)
Reduced body weight development in the dams during pregnancy and lactation
- Average weight gain 61 % less than the control between days 0-20 of pregnancy
- Mean body weight on day 20 of pregnancy 24% below control
- Body weights persisted being 19-23% below control during lactation days 0-4, though weight gain was about 60% above control in these females after cessation of exposure on gestation day 18

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of the high concentration animals was either decreased only marginally and of transient matter, or increased slightly compared to the control.

Food efficiency:

no effects observed

Description (incidence and severity):

The food efficiency of the high concentration animals was, when compared with the control, only reduced transiently at the beginning of the exposure (day 9).

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There are no treatment-related changes in the haematological parameters measured. Clotting analysis revealed prolonged prothrombin times in the blood of the males of the high concentration group at the end of the study.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Regarding clinical pathology findings, in the high concentration group females, indications of a reduced general state was seen, characterized by significantly decreased serum creatinine, total protein, albumin and globulin levels. No treatment-related effects were observed in the clinical pathology parameters of the animals of the low and mid concentration groups.

Summary of the treatment-related findings, test group 3 (0.956 mg/l = 180 ppm): :
-Increased prothrombin times, urea*, magnesium*, urinary specific gravity and urinary casts* in the males
-Decreased triglycerides** and urinary volume in the males
-Decreased chloride, creatinine*, total bilirubin, total protein**, albumin* and globulins** in the females
(* statistically significant to a level of 95 %
** statistically significant to a level of 99 %)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Regarding clinical pathology findings, treatment-related effects were observed only in the high concentration groups. The investigations revealed mild impairment of renal function in the males, substantiated by slightly increased urea concentrations in the serum, excretion of decreased urinary volume with increased specific gravity and the presence of urinary casts.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment related findings were observed in the functional observation battery and motor activity examinations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology detected treatment-related hyperplasia of the respiratory epithelium in the anterior part of the nasal cavity (cut level I) of males (higher incidence) and of females (higher grades of severity). One male of the high dose group showed hyperplasia of the respiratory epithelium also in cut level III of the nasal cavity.
Hyperplasia of the respiratory epithelium in the nasal cavity was interpreted as an adaptive, reversible reaction to the inhaled test article.

All other microscopic findings recorded were either single observations, or they occurred in control animals only, or they were recorded at low or at comparable incidence and graded severity in control and high dose males and/or females. Hence, they were all regarded to have developed fortuitously and unrelated to treatment.

Summary of the treatment-related findings, test group 3 (0.956 mg/l = 180 ppm): :
Hyperplasia of the respiratory epithelium in the nasal cavity at level I in male (incidence) and in female rats (graded severity, only)
Hyperplasia of the respiratory epithelium in the nasal cavity at level III in one male rat.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Male cohabitation data
For all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed. Thus, the male mating index reached 100% in all groups, including the controls.
Fertility could be proven for nearly all F0 parental males within the scheduled mating interval for F1 litter. One mid dose male and one high dose male did not generate F1 pups. Thus, the male fertility index ranged between 89% and 100%. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. For none of the affected male rats conclusive histopathological findings were gathered which could account for the observed impaired fertility.

- Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 100% for all groups.
The mean cohabitation time (duration until sperm was detected (i.e. day 0 p.c.)) amounted to 3.3 days/2.3 days/2.5 days/2.0 days (0, 0.106, 0.319 and 0.956 mg/l). These values reflect the normal range of biological variation inherent in the strain used in this study. Consequently, the differences between the groups are assessed as spontaneous in nature and without any biological relevance.
All sperm positive rats delivered pups with the following exceptions: one mid dose F0 parental female and one high dose female did not become pregnant. Therefore, the fertility indices ranged between 89% and 100%.

The mean duration of gestation was very similar in the test groups 0-3 (between 21.8 and 21.9 days).

The gestation index was 100% in test groups exposed to 0, 0.106 and 0.319 mg/l, but was markedly reduced to 67% in the high dose group (0.956 mg/L). Only 4 out of 6 surviving presumed pregnant females had liveborn pups in the high dose group.

The number of liveborn and stillborn pups was comparable between the control and test groups 1 and 2, while there were a statistically significantly decreased number of liveborn and an increased number of stillborn pups in test group 3. Thus, the live birth index amounted to 100% in test groups exposed to 0, 0.106 and 0.319 mg/l and 80% in test group 3 (0.956 mg/L).

Dose group (mg/L): 0 - 0.106 - 0.319 - 0.956
cohabitation time (days): 3.3 - 2.3 - 2.5 - 2.0
male mating index (%) : 100 - 100 - 100 - 100
male fertility index (%): 100 - 100 - 90 - 89
female mating index (%): 100 - 100 - 100 - 100
female fertility index (%) 100 100 90 89
duration of gestation (days) 21.8 21.9 21.9 21.8
gestation index (%) 100 100 100 67
pups delivered 11.0 9.7 10.4 8.3
live birth index (%) 100 100 100 80**
Stillborn (%) 0 0 0 20**

(** significant to a level of 99 %)

Key result

Dose descriptor:

NOAEC

Effect level:

0.319 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

urinalysis

histopathology: non-neoplastic

Remarks on result:

other: marked maternal toxicity, including mortality

Key result

Dose descriptor:

NOAEC

Effect level:

0.319 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Test group 3 (956 mg/m3 ): markedly lower viability index (20% vs 97% in the control)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test group 3 (956 mg/m3 ): lower pup body weights on day 1 p .p . (28% below control ). Average pup body weights and body weight gain significantly below control (42% on
day 4 p.p. and 67% for days 1-4 p.p., respectively)

Sexual maturation:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.319 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Key result

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

0.956 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Reference 2

Endpoint:

reproductive toxicity, other

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Please find the attached justification.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no reproductive or systemic effects observed

Remarks on result:

other: Result read-across source CAS No. 141-32-2.

Remarks:

Correction for molecular weight is not required.

Dose descriptor:

NOEC

Effect level:

ca. 0.4 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Result read-across source CAS No. 96-33-3, corrected for molecular weight.

Dose descriptor:

NOEC

Effect level:

ca. 0.028 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Result read-across source CAS No. 96-33-3, corrected for molecular weight.

Dose descriptor:

NOAEC

Effect level:

ca. 0.522 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Result read-across source CAS No.103-11-7, corrected for molecular weight.

Dose descriptor:

NOAEC

Effect level:

0.522 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Result read-across source CAS No.103-11-7, corrected for molecular weight.

Dose descriptor:

NOAEC

Effect level:

0.157 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Remarks on result:

other: Result read-across source CAS No.103-11-7, corrected for molecular weight.

Dose descriptor:

NOAEC

Effect level:

2.86 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Result read-across source CAS No. 141-32-2.

Remarks:

Correction for molecular weight is not required.

Dose descriptor:

NOAEC

Effect level:

0.57 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

Remarks on result:

other: Result read-across source CAS No. 141-32-2.

Remarks:

Correction for molecular weight is not required.

Dose descriptor:

NOAEC

Effect level:

0.11 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Result read-across source CAS No. 141-32-2.

Remarks:

Correction for molecular weight not required.

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOEC

Effect level:

ca. 0.4 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Result read-across source CAS No. 96-33-3, corrected for molecular weight.

Key result

Dose descriptor:

NOEC

Effect level:

0.028 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Result read-across source CAS No. 96-33-3, corrected for molecular weight.

Key result

Critical effects observed:

no

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Result read-across source CAS No. 141-32-2 and 103-11-7

Dose descriptor:

NOEC

Generation:

F1

Effect level:

ca. 1.37 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Result read-across source CAS No. 96-33-3, corrected for molecular weight.

Key result

Critical effects observed:

no

Dose descriptor:

NOEC

Generation:

F2

Effect level:

0.137 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Result read-across source CAS No. 96-33-3, corrected for molecular weight.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Study according to OECD TG 443, GLP

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

319 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Study according to OECD TG 413/422, GLP

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Tert-butyl acrylate was tested in a combined sub-chronic toxicity study with a reproduction / developmental toxicity screening test by inhalation in rats (based on OECD-guidelines 413 and 422) (BASF 2004). Groups of ten male and ten female Wistar rats were exposed to vapours of tert-butyl acrylate for 6 hours/day and 5 days/week to concentrations of 20, 60 and 180 ppm (corresponding to 0.106, 0.319 and 0.956 mg/L). A concurrent control group was exposed to clean air. The males were treated for approx. 13 weeks (10 weeks premating, 3 weeks mating and post mating). In females treatment lasted from 10 weeks premating, during mating and gestation through day 4 after delivery (approx. 15 weeks). No effects were seen in the low (20 ppm) and mid (60 ppm) dose group. Due to the significant decrease of the mean terminal body weight in animals (males -17.4 %; females - 9.4 %) of the high dose group (180 ppm), some mean relative organ weights were increased, e.g. the relative testes and epididymis weight of males. No morphologic alterations were noted in those organs. For all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed. Thus, the male mating index reached 100% in all groups, including the controls. One mid dose male and one high dose male did not generate F1 pups. Thus, the male fertility index ranged between 89% and 100%. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The female mating index calculated after the mating period for F1 litter was 100% for all groups. The gestation index was markedly reduced to 67% in the high dose group, only 4 out of 6 surviving presumed pregnant females had liveborn pups. The number of liveborn and stillborn pups, the live birth index amounted to 100% in test groups exposed to 0, 20 and 60 ppm and 80% in the high dose group (180 ppm). Thus, the NOAEC for fertility of the parent animals was determined to be 0.319 mg/L (60 ppm).

In addition, there are data from the structural analogue (see read-across justification) methyl acrylate.

In a two-generation study according to OECD TG 416 groups of 27 male and female Crl:CD(SD) rats were whole-body exposed to methyl acrylate vapours at target concentrations of 0, 5, 25, and 75 ppm for six hours/day, seven days/week, resulting in actual average concentrations of 0, 5.3 ± 0.2, 25.7 ± 0.3, and 75.4 ± 0.6 ppm, respectively (corresponding to approx. 0, 0.019, 0.092, and 0.269 mg/L). (BAMM2009) Rats were exposed daily for approximately ten weeks prior to breeding, and continuing through breeding, gestation and lactation for two generations. Maternal rats were not exposed after GD 20 through LD 4 in order to allow for parturition and initiation of lactation. Exposure of maternal rats continued from LD 5 – LD 28. In-life parameters included clinical observations, feed consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. In addition, post-mortem evaluations included gross pathology, histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.

Treatment-related effects in parental rats exposed to 75 ppm included decreased body weight and feed consumption in males and females throughout most of the two generation study. There were no effects on body weight or feed consumption at 25 or 5 ppm. Treatment-related, adverse histopathologic effects were present in the nasal tissues of P1 and P2 males and females exposed to 25 or 75 ppm. The incidence and severity of the nasal effects were concentration dependent. Degeneration with regeneration of the olfactory epithelium (very slight to moderate) occurred in all P1 and P2 males and females exposed to 75 ppm. Very slight olfactory epithelial degeneration, without accompanying regenerative hyperplasia, was noted in some of the P1 and P2 females and P2 males exposed to 25 ppm. There were several histopathologic effects that accompanied the degeneration of the olfactory epithelium. Very slight or slight degeneration of the olfactory nerve was present in most of the P1 and P2 males and females exposed to 75 ppm, and one P1 male exposed to 25 ppm. Very slight or slight chronic-active inflammation was present in 16/27 P1 males, 20/27 P1 females, 14/27 P2 males, and 8/27 P2 females exposed to 75 ppm, and in one or two males and females from both generations exposed to 25 ppm. Very slight necrosis of individual olfactory epithelial cells was present in most of the P1 and P2 males and females exposed to 75 ppm, and a few P1 and P2 animals (one to four per sex) exposed to 25 ppm. Very slight mineralization of the olfactory epithelium was present in one or two P1 and P2 animals exposed to 25 ppm, and in 6/27 P1 males, 4/27 P1 females, 16/27 P2 males and 14/27 P2 females exposed to 75 ppm. Other nasal effects consisted of an increase in the incidence of very slight or slight hyperplasia of the transitional epithelium in P1 and P2 males and females exposed to 25 or 75 ppm, and an increase in the incidence of very slight or slight hyperplasia and hypertrophy of the respiratory epithelium in P1 males and females exposed to 25 or 75 ppm, and in P2 males and females exposed to 75 ppm. There were no treatment-related histopathologic effects in P1 or P2 animals exposed to 5 ppm. No treatment-related effects were seen in reproductive function or pup survival. However, pup body weights of the 75 ppm exposure group were decreased on postnatal day 14-28 in both generations. There were no effects on pup body weight in rats exposed to 25 or 5 ppm. The effects on pup body weight, as well as the changes in parental body weight and feed consumption, likely were secondary changes all stemming from nasal irritation and resultant stress.

In summary, the no-observed-adverse-effect concentration (NOAEC) for parental systemic toxicity was determined to be 5 ppm (= ca. 0.019 mg/L) and was based on histologic changes in the nasal tissues seen at higher concentrations. The NOAEC for developmental toxicity was 25 ppm (= ca. 0.092 mg/L), based on decreases in pup body weight at 75 ppm which were secondary to parental toxicity. The NOAEC for reproductive toxicity was 75 ppm (= ca. 0.269 mg/L), the highest concentration tested. After correction for molecular weight, the effect values for tert-butyl acrylate are determined at 0.028 mg/L (NOAEC parental systemic toxicity), 0,137 mg/L (NOAEC for developmental toxicity) and 0.4 (NOAEC for reproductive toxicity, the highest concentration tested.)

In addition, there are data from the structural analogue (see read-across justification) n-butyl acrylate.

For n-butyl acrylate an extended one generation study according to OECD 443 and GLP was performed. 30 Crl:CD(SD) rats were exposed to 20, 50 and 150 mg/kg bw/day by oral (gavage) exposure route (Acrylate Reach TF, 2017).

There were no test substance-related effects on survival for F0 and F1 animals at any dosage level. No test substance-related clinical observations were noted for F0 and F1 animals at any dosage level. Mean body weights, body weight gains, food consumption, and food efficiency in the 20, 50, and 150 mg/kg/day F0 and F1 males and females were unaffected by test substance administration. No test substance-related effects were noted on F0 reproductive performance (male and female mating and fertility, male copulation, and female conception indices), the mean number of days between pairing and coitus, mean gestation lengths, or the process of parturition. In addition, there were no test substance-related effects on F0 or F1 estrous cyclicity or spermatogenic parameters (testicular and epididymal sperm concentrations, sperm production rate, sperm motility, and sperm morphology) at any dosage level.

There were no test substance-related effects on the number of F1 pups born, live litter size, percentage of males at birth, F1 postnatal survival, clinical observations, anogenital distance,

offspring body weights, necropsy findings, or developmental landmarks (areolae/nipple retention, vaginal patency, and balanopreputial separation).

No test substance-related effects on clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were noted for F0 and F1 animals at any dosage level. In addition, no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroid stimulating hormone) were noted in F0 and F1 males or females or F1 pups (on PND 4 and 21). Test substance-related histologic changes were observed in all dosage groups in the F0 generation and F1 males and females in Cohort 1A. Epithelial hyperplasia and/or hyperkeratosis was observed in the nonglandular stomach in all test substance-treated groups examined. Mild to moderate changes in the 150 mg/kg/day group males and females of the F0 and F1 generations were considered adverse in this study. Microscopic changes in the stomach were associated with the gross observation of thickened nonglandular stomach, but were not associated with any clinical pathology, organ, or body weight changes. In the F0 generation, a nonadverse increased incidence of biliary hyperplasia (males and females) and random hepatocellular necrosis (males) were observed in the liver in the 150 mg/kg/day group. Additionally, nonadverse test substance-related microscopic findings (increased severity of mineralization at the

corticomedullary junction) were observed in the kidneys of the 150 mg/kg/day group F0 females. Thickened stomachs were noted in the 50 and 150 mg/kg/day group F1 males and in the 150 mg/kg/day group F1 females at the scheduled necropsies for Cohort 1B; this finding was considered test substance-related and adverse in the 150 mg/kg/day group males and females. No other test substance-related internal findings were observed at any dosage level for F1 Cohort 1B animals. No test substance-related effects on the mean number of F0 implantation sites or number of unaccounted-for sites were noted at any dosage level. No test substance-related macroscopic findings were observed in F1 pups that were found dead, culled on PND 4, or examined at the scheduled necropsy on PND 21; F1 pup organ weights on PND 21 were unaffected by test substance administration. No test substance-related effects on ovarian primordial follicle counts were noted in the F0 females suspected of reduced fertility or F1 Cohort 1A females. There were no test substance-related effects on organ weights noted for F0 and F1 males and females at any dosage level.

Due to the absence of systemic toxicity noted for F0 and F1 males and females throughout the study, a dosage level of 150 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 and F1 male and female systemic toxicity when n-butyl acrylate was administered orally by gavage to Crl:CD(SD) rats. Epithelial hyperplasia and/or hyperkeratosis in the nonglandular stomach noted in the 150 mg/kg/day group F0 and F1 males and females were considered adverse; based on these results, 50 mg/kg/day was considered to be the NOAEL and 150 mg/kg/day was considered to be the lowest-observed-adverse-effect level (LOAEL) for local effects in the F0 and F1 generations. Based on the lack of effects noted for F1 litters, a dosage level of 150 mg/kg/day was considered to be the NOAEL for neonatal toxicity. There was no evidence of reproductive toxicity at any dosage level based on evaluation of reproductive performance in the F0 generation and sperm measurements and estrous cyclicity in the F0 and F1 generations. Therefore, the NOAEL for F0 and F1 reproductive toxicity was considered to be 150 mg/kg/day.

In an OECD 413 study in Sprague Dawley rats (20 animals per sex and dose) were exposed by inhalation to measured concentrations of 0, 21, 108, 211 and 546 ppm n-butyl acrylate (corresponding to approx. 0, 0.11, 0.57, 1.11, 2.86 mg/L) for 6 hours per day, 5 days/week for 13 weeks (BASF 1978). Clinical, clinico-chemical, haematological, gross-pathological, and histopathological examinations revealed no substance-related effects in the 21 and 108 ppm dose groups. At 211 ppm, the test substance caused eye irritation and irritation of the nasal mucosa. Significant reductions in body weight changes (13.3 %) were observed. In clinico-chemical examinations of females, decreased potassium values and an increase in alkaline phosphatase activity were observed. In the 546 ppm dose group, 31 of 40 animals (77 %) died. Hemorrhagic discharge from eyes and noses and severe dyspnoea were observed, which became constantly more severe. Many clinico-chemical and haematological parameters were affected in animals of this dose group. The animals died during exposure due to strong irritation of the respiratory tract. Metaplasia of the respiratory epithelium as far as the terminal bronchioles and proliferation of the bronchoalveolar epithelium could be detected in histopathological examinations. The NOAEC for this study is 108 ppm(0.57 mg/L/day) and the LOAEC is 211 ppm (1.11 mg/L/day) based on body weight decrease, clinico-chemical changes, and changed organ weights. The NOAEC for local effects (histological changes in the nasal mucosa and olfactory epithelium) is 21 ppm (0.11 mg/L/day) and the LOAEC is 108 ppm (0.57 mg/L/day).No substance-related effect could be determined by organ weight determinations, gross - and histopathology in the reproductive organs of the male and female animals at any concentration. The NOAEC for effects on reproductive organs was 546 ppm (2.86 mg/L), the highest concentration investigated.

Conclusion:

Based on the data derived from the combined sub-chronic toxicity study with a reproduction / developmental toxicity screening test, tert-butyl acrylate is not expected to impair reproduction. Also a two-generation study with the structural analogue methyl acrylate did not give any indications for a fertility impairing effect. This is supported by an extended one generation, subchronic and chronic studies with the structural analogue n-butyl acrylate, in which no effects to the gonads could be determined. Overall, the data on t-butyl acrylate itself as well as from the read-across substances methyl acrylate and n-butyl acrylate gives no indications that this class of chemicals has fertility impairing effects.

In analogy to nBA the DNEL for tBA is based on the OEL of nBA of 2 ppm. Using the DNEL derivation according to the ECHA Guidance on information requirements and chemical safety assessment, Chapter R.8: Characterization of dose (concentration)-response for human health supports the OEL also for tBA). The German MAK Commission and the EU Scientific Committee on Occupational Exposure Limits (SCOEL) derived the OEL of 2 ppm which was taken as DNEL based on the most sensitive endpoint the local irritation of the olfactory epithelium of the nasal mucous membranes which occurred in the 2 year inhalation study with n-butyl acrylate at 15 ppm. Based on the above mentioned studies, not indicating any suspicion for a reproductive toxic effect in much higher concentrations, a further animal study will not provide more safety to workers.

Effects on developmental toxicity

Description of key information

Tert. butyl acrylate was tested in a prenatal developmental toxicity study according to OECD TG 414 in rats by oral gavage. There were no adverse effects on maternal survival, clinical observations, body weights, food consumption, and gross necropsy or microscopic findings at any dosage level. Based on these results, a dosage level of 120 mg/kg/day was considered to be the no‑observed‑adverse‑effect level (NOAEL) for maternal toxicity when t‑butyl acrylate was administered orally by gavage to time-mated Crl:CD(SD) rats. Mild epithelial hyperplasia and/or hyperkeratosis in the nonglandular stomach noted in the mid and high dose level, therefore the NOEL for local effects was set at 30 mg/kg/day. Intrauterine growth and survival and fetal morphology (external, visceral, and skeletal) were unaffected by test substance administration at all dosage levels. Based on these results, a dosage level of 120 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity. (BASF 2017)

A combined sub-chronic toxicity study with a reproduction / developmental toxicity screening test with tert-butyl acrylate did not reveal any potential of the substance to cause a developmental toxic / teratogenic effect. This is supported by data on the structural analogues n-butyl acrylate, methyl acrylate, ethyl acrylate and 2-ethylhexyl acrylate.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Aug 2002 - 04 Dec 2003 (experimental)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 22, 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

May 12, 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental)

Version / remarks:

712-C-00-368; July, 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

712-C-98-204; August, 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC L133 (Sub-Chronic Inhalation Toxicity Study)

Version / remarks:

May 30, 1988

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Tert.- Butylacrylat
- Physical state: colorless liquid
- Analytical purity: 99.62% (Batch No. 2-011002-15/1) - 99.8% (Batch No. B602)
- Purity test report: Analytical report 02L00206 (Batch No. B602); reanalysis (Batch No. 2-011002-15/1)
- Stability under test conditions: The stability under storage conditions over the exposure period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: refrigerator, protected from light and stored under air

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: ca. 4 weeks
- Weight at study initiation: Males: 106.1-108.3 g; Females: 92.7-94.7 g (groupwise)
- Housing: individually in type DK II stainless steel wire mesh cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Underneath the DK III cages, waste trays were fixed containing bedding material (type 3/4 dust free embedding (SSNIFF, Soest, FRG); from day 18 post coitum until sacrifice, the pregnant animals and their litters were housed in Markolon M III cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Pregnant females were provided with nesting material (cellulose wadding) towards the end of pregnancy.
- Diet: ad libitum, "GLP" (Provimi Kliba SA, Kaiseraugst, CH)
- Water: ad libitum, tap water
- Acclimation period: ca. 2 weeks

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

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: clean air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Piston metering pumps (Sarstedt DESAGA) and glass vaporizers with thermostat (BASF AG
- Method of holding animals in test chamber: The animals were kept singly in wire cages located in a glass-steel inhalation chamber, volume of 1.4 m3 (BASF AG).
- Method of conditioning air: For each concentration the test substance was supplied to a thermostated vaporizer at a constant rate by means of the metering pump. The vapor was generated with conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C) and passed into the inhalation system.
- Temperature, pressure in air chamber: 25 ± 3°C, -10 Pa
- Air flow rate: 27.5-28.5 m3/h

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were analyzed by gas chromatography in all test groups including control (Hewlett-Packard 5840 A). Daily means were calculated based on 2 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. The concentration constancy in each inhalation system was continuously monitored by means of a total hydrocarbon analyzer.
To ensure, that no liquid aerosols were formed at concentration levels as high as 180 ppm, a scattered light photometer was used to monitor the test atmosphere of the high dose group.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Target concentrations were: 0.106, 0.319, and 0.956 mg/L (corresponding to 20, 60, and 180 ppm)
- Measured concentrations were: 0.107 ± 0.0061, 0.317 ± 0.0211, 0.958 ± 0.0481 mg/L

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: from study day 69 - study day 80
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

- males: ca. 13 weeks (10 weeks premating, 3 weeks mating and post mating)
- females: ca. 15 weeks (10 weeks premating, during mating and gestation through day 4 after delivery)

Frequency of treatment:

6 hours/day, 5 days/week

Duration of test:

- males: ca. 13 weeks (10 weeks premating, 3 weeks mating and post mating)
- females: ca. 15 weeks (10 weeks premating, during mating and gestation through day 4 after delivery)

Dose / conc.:

0.106 mg/L air (nominal)

Remarks:

equals 20 ppm

Dose / conc.:

0.319 mg/L air (nominal)

Remarks:

equals 60 ppm

Dose / conc.:

0.956 mg/L air (nominal)

Remarks:

equals 180 ppm

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
106 mg/m3 (ca. 20 ppm) : as the expected no observed adverse effect level
319 mg/m3 (ca. 60 ppm) : as the intermediate dose level
956 mg/m3 (ca. 180 ppm): as the dose level where toxic effects were expected

Preflow period of 4 days

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at least 3 times (before, during and after exposure) on exposure days and once during the preflow period, on the day of neurofunctional test and prior to gross necropsy. During exposure only a group wise examination was possible. The nesting, littering, and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. The littering behavior of the dams was also inspected on each workday in the afternoons in addition to the evaluations in the mornings.

BODY WEIGHT:
- Time schedule for examinations: day -7, on day -4 (start preflow period), on day 0(start exposure period) and then in weekly intervals as well as prior to gross necropsy.

FOOD CONSUMPTION:
- Time schedule: day -7, on day -4 (start preflow period), on day 0 (start of exposure period) and then in weekly intervals.
- It was calculated as mean food consumption in grams per animal and day.
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

HAEMATOLOGY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular, haemoglobin concentration, platelets, differential blood count, prothrombin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium, bile acids

GROSS PATHOLOGY:
The animals were sacrificed under narcoren® anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology. To prevent post mortem autolysis, the animals that died intercurrently were necropsied as soon as possible after death.

ORGAN WEIGHTS:
The following weights parameters from all animals sacrificed were determined: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, uterus, thymus, spleen, brain, heart, lungs

HISTOPATHOLOGY:
-The following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, spinal cord (cervical, thoracic and lumber cord), sciatic nerve, pituitary gland, salivary glands (glandula mandibularis and glandula sublingualis), thyroid glands/parathyroid glands, adrenal glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes (mandibular and mesenteric), spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, oesophagus, stomach (forestomach and glandular stomach), duodenum, jejunum ileum, caecum, colon, rectum, urinary bladder, sternum with marrow, bone marrow (femur), skull (with nasal cavities, larynx, pharynx, eyes with optic nerve, femur with knee joint, skin, skeletal muscle, extraorbital lacrimal glands. Ovaries of animals that were killed as scheduled were fixed in Bouin's solution and embedded in paraplast, thereafter. Ovaries of animals that died intercurrently were fixed in 4% formaldehyde solution.
- After the organs were fixed, histotechinical processing and examination was were performed as follows: Nasal cavities (level I- IV), Larynx (level I- III), Trachea (longitudinal, with carina), Lungs (5 lobes) and thyroid glands/parathyroid glands in all animals; all gross lesions in all affected animals; evaluations of all other organs and tissues fixed were only performed in animals of the control and high dose group

NEUROBEHAVIOURAL EXAMINATION:
- Detailed clinical observation (DCO) were performed in all animals prior to the exposure period and thereafter in weekly intervals. The findings were ranked according to the degree of severity, if applicable. The following parameters were examined: abnormal behaviour during handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, faeces (appearance/consistency), urine, pupil size
- A functional observational battery (FOB) was carried out on the assigned animals (5 males and 5 females/ test group) on study days 56 and 57 for males and females, respectively. On the days of neurofunctional tests there was no exposure of the concerning animals as well as the other 5 animals of the same test group. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. During the home cage observations attention was paid to posture, tremor, convulsions, abnormal movements, impairments of gait and other findings. During the open field observations the following parameters were examined: behaviour when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, faeces (number of faecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within 2 minutes and other findings. After the open field test animals were subjected to the following sensorimotor or reflex tests: approach response, touch response, vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behaviour during “handling”, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test and other finding. All findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Motor activity (MA) was measured on the same day and with the same animals as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order . The measurements started at about 14:00 p.m. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages ; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

All surviving pups (after sacrifice on day 4 p.p. by means of C02), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

Two-sided Dunnett test for food consumption, body weight and body weight change, number of mating days, duration of gestation, number of pups
delivered per litter.
Pairwise comparison by the Fisher´s exact test for male and female mating index, male and female fertility index, 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 and urine analysis except vlume, color, turbidity and specific gravity.
Pairwise comparison by the Wilcoxon test for the proportions of affected pups per litter with necropsy observations.
Non-parametric Kruskal-Wallis test (two-sided)/Wilcoxon test for feces, rearing, grip strength forelimbs, grip strength hindlimbs, landing foot-splay test, motor activity for the different time intervals, clinical pathology parameters except differential blood count and organ weights.

Indices:

- Viability index (%) = number of live pups on day 4 after birth / number of liveborn pups on the day of birth x 100
- Sex ratio (%) – number of live male or female pups on day (0/4) / number of live male and female pups on day (0/4) x 100

Historical control data:

not reported

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Unspecific clinical symptoms indicative for some irritation and systemic toxicity in the high dose group (comprising visually increased respiration, salivation, piloerection, eyelid closure, eye discharge, alopecia, aggressiveness, hyperactivity and apathy).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Two female animals exposed to the high concentration died on study day 88 and 91 (day 18 and 20 of gestation), respectively. Both animals were found pregnant at death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test group 3 (0.956 mg/L = 180 ppm):

Retarded body weight development of the females
- mean body weight: - 3.0 % and -8.0 % of the control from study day 9 onward
(statistically significant on study day 51 )
- mean body weight gain: -19.9 % to - 41.8 % of the control from study day 9 onward (statistically significant )

- Decreased mean terminal body weight in females.

Reduced body weight development in the dams during pregnancy and lactation
- Average weight gain 61 % less than the control between days 0-20 of pregnancy
- Mean body weight on day 20 of pregnancy 24% below control
- Body weights persisted being 19-23% below control during lactation days 0-4, though weight gain was about 60% above control in these females after cessation of exposure on gestation day 18.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of the high concentration animals was either decreased only marginally and of transient matter, or increased slightly compared to the control.
Slightly to moderately reduced food consumption of dams during pregnancy (about 16% below control) and lactation days 0-4 (about 39% below control)

Food efficiency:

no effects observed

Description (incidence and severity):

The food efficiency of the high concentration animals was, when compared with the control, only reduced transiently at the beginning of the exposure (day 9).

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There are no treatment-related changes in the haematological parameters measured.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Regarding clinical pathology findings, in the high concentration group females, indications of a reduced general state was seen, characterized by significantly decreased serum creatinine, total protein, albumin and globulin levels. No treatment-related effects were observed in the clinical pathology parameters of the animals of the low and mid concentration groups.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment related findings were observed in the functional observation battery and motor activity examinations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Due to the significant decrease of the mean terminal body weight in animals (females 9,4 %) of the high dose group, some mean relative organ weights were increased. No morphologic alterations were noted in those organs.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Two female animals of the high dose group died prematurely, revealing non-specific organ changes (atrophy) in the lymphoid tissues of spleen and thymus (both animals) or prefinal erosion/ulcer in the mucosa of the glandular stomach (one animal). Although these findings rather reflect the consequence of a longer story of illness than a treatment-related effect and although none of the findings in thymus, spleen and/or glandular stomach were recorded in the animals killed at scheduled dates.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hyperplasia of the respiratory epithelium in the nasal cavity at level I in male (incidence) and in female rats (graded severity, only) Hyperplasia of the respiratory epithelium in the nasal cavity at level III in one male rat.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

In high dose group lower live birth index (80% vs 100% in the control). Lower gestation index (67% vs 100% in the control), 4 out of 6 surviving dams with
liveborn pups, 2 dams with only stillborn pups

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Insufficient maternal care in 3 females of the high dose group with subsequent complete litter loss until day 3 post delivery

Key result

Dose descriptor:

NOAEC

Effect level:

0.319 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: non-neoplastic

mortality

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

High dose group: Lower pup body weights on day 1 p.p. (28% below control). In addition, average pup body weights and body weight gain significantly below control (42% on day 4 p.p. and 67% for days 1-4 p.p., respectively)

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

In high dose group: lower live birth index (80% vs 100% in the control ). Lower gestation index (67% vs . 100% in the control), 4 out of 6 surviving dams with
liveborn pups, 2 dams with only stillborn pups

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

High dose group: markedly lower viability index (20% vs 97% in the control). Insufficient maternal care in 3 females of the high dose group with subsequent complete litter loss until day 3 post delivery.

External malformations:

no effects observed

Description (incidence and severity):

At pup necropsy, no malformations were found at any dose level.

Skeletal malformations:

no effects observed

Description (incidence and severity):

At pup necropsy, no malformations were found at any dose level.

Visceral malformations:

no effects observed

Description (incidence and severity):

At pup necropsy, no malformations were found at any dose level.

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
At the highest dose level (0.956 mg/L) the pre- and postnatal development of the offspring was substantially impaired. At test concentrations where no maternal toxicity was observed, no embryotoxic,

Key result

Dose descriptor:

NOAEC

Effect level:

0.319 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

changes in postnatal survival

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

0.956 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:

yes

Dose group  (mg/L)           0     0.106   0.319    0.956
maternal body weight 
change gd 0 - 20 (g)         91.6  91.5    90.2     35.7**
maternal body weight
lactation day 0 - 4 (g)      10    10      9        6 
maternal mortality during 
gestation (%)                0     0       0        20      
duration of gestation (days) 21.8  21.9    21.9     21.8
gestation index (%)          100   100     100      67
pups delivered               11.0  9.7     10.4     8.3
live birth index (%)         100   100     100      80**
Stillborn (%)                0     0       0        20**    

pups died (%)                0.9   0       1.1      36**
pups cannibalized (%)        1.8   1.0     0        28**
pups viability index         97    99      99       20**
pups weight day 1 (g)        6.0   6.2     6.2      4.3**
pups weight day 4 (g)        8.9   9.0     9.2      5.2**

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

Please find the attached justification.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

rabbit oral

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal systemic toxicity & maternal developmental toxicity

Remarks on result:

other: Result read-across source CAS No. 141-32-2.

Remarks:

Correction for molecular weight is not required.

Dose descriptor:

NOAEL

Remarks:

rat oral

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No reproductive or systemic effects observed

Remarks on result:

other: Result read-across source CAS No. 141-32-2.

Remarks:

Correction for molecular weight is not required.

Dose descriptor:

NOAEC

Remarks:

rabbit inhalation

Effect level:

0.082 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Result read-across source CAS No. 96-33-3, corrected for molecular weight.

Dose descriptor:

NOAEC

Remarks:

rat inhalation

Effect level:

ca. 0.13 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Remarks on result:

other: Result read-across source CAS No. 141-32-2.

Remarks:

Correction for molecular weight is not required.

Dose descriptor:

NOAEC

Remarks:

rat inhalation

Effect level:

ca. 0.13 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Remarks on result:

other: Result read-across source CAS No. 141-32-2.

Remarks:

Correction for molecular weight is not required

Dose descriptor:

LOAEC

Remarks:

rat inhalation

Effect level:

ca. 0.52 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Result read-across source CAS No. 141-32-2.

Remarks:

Correction for molecular weight is not required

Dose descriptor:

NOAEC

Remarks:

rat inhalation

Effect level:

ca. 0.525 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Result read-across source CAS No.140-88-5, corrected for molecular weight.

Dose descriptor:

NOAEC

Remarks:

rat inhalation

Effect level:

0.269 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Result read-across source CAS No.140-88-5, corrected for molecular weight.

Dose descriptor:

NOAEC

Remarks:

rat inhalation

Effect level:

ca. 0.39 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Result read-across source CAS No.103-11-7, corrected for molecular weight.

Dose descriptor:

NOAEL

Remarks:

mice oral

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

mortality

Remarks on result:

other: Result read-across source CAS No. 141-32-2.

Remarks:

Correction for molecular weight is not required.

Key result

Abnormalities:

no effects observed

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

CAS No.141-32-2 (rabbit oral, rat oral, rat inhalation), CAS No. 96-33-3 (rabbit inhalation), CAS No. 103-11-7 (rat inhalation)

Dose descriptor:

NOAEC

Remarks:

rat inhalation

Effect level:

ca. 0.52 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

other: Result read-across source CAS No. 141-32-2.

Remarks:

Correction for molecular weight is not required.

Dose descriptor:

NOAEC

Remarks:

rat inhalation

Effect level:

0.525 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

other: Result read-across source CAS No.140-88-5, corrected for molecular weight

Dose descriptor:

NOAEL

Remarks:

mice oral

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

other: Result read-across source CAS No. 141-32-2. Correction for molecular weight is not required

Dose descriptor:

NOAEL

Remarks:

mice oral

Effect level:

2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

Remarks on result:

other: Result read-across source CAS No. 141-32-2. Correction for molecular weight is not required

Key result

Abnormalities:

no effects observed

Abnormalities:

effects observed, treatment-related

Localisation:

external: eye

external: face

skeletal: sternum

skeletal: rib

other: exencephaly

Description (incidence and severity):

Source CAS No. 141-32-2 (mice oral)

Key result

Developmental effects observed:

no

Developmental effects observed:

yes

Lowest effective dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Developmental effects observed:

yes

Lowest effective dose / conc.:

0.71 mg/L air (analytical)

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:

not specified

Developmental effects observed:

yes

Lowest effective dose / conc.:

1.05 mg/L air (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:

not specified

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 500 mg/kg bw/day (actual dose received)

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:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study according to OECD TG 414, GLP

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Tert. butyl acrylate was tested in a prenatal developmental toxicity study according to OECD TG 414 in rats by oral gavage. The dose selelction of 30, 60, and 120 mg/kg/day was done based on a maternal range-finding study (higher doses caused mortality).

All females in the control, 30, 60, and 120 mg/kg/day groups survived to the scheduled necropsy. No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. Mean maternal body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 30, 60, and 120 mg/kg/day groups were unaffected by test substance administration.

At the scheduled necropsy on gestation Day 21, no gross observations were considered to be associated with administration of the test substance.

Test substance-related squamous epithelium hyperplasia and hyperkeratosis of the nonglandular stomach were observed in the 30, 60, and 120 mg/kg/day groups. These microscopic findings ranged from minimal to mild and increased in severity with increasing dosage levels, but were not considered adverse. No other test substance-related microscopic findings were noted.

Intrauterine growth and survival were unaffected by test substance administration. In addition, no test substance-related external, visceral, and skeletal malformations and developmental variations were noted at any dosage level.

There were no adverse effects on maternal survival, clinical observations, body weights, food consumption, and gross necropsy or microscopic findings at any dosage level. Based on these results, a dosage level of 120 mg/kg/day was considered to be the no‑observed‑adverse‑effect level (NOAEL) for maternal toxicity when t‑butyl acrylate was administered orally by gavage to time-mated Crl:CD(SD) rats. Mild epithelial hyperplasia and/or hyperkeratosis in the nonglandular stomach noted in the mid and high dose level, therefore the NOEL for local effects was set at 30 mg/kg/day. Intrauterine growth and survival and fetal morphology (external, visceral, and skeletal) were unaffected by test substance administration at all dosage levels. Based on these results, a dosage level of 120 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity. (BASF 2017)

Tert. butyl acrylate was tested in a combined sub-chronic toxicity study with a reproduction / developmental toxicity screening test by inhalation in rats (based on OECD-guidelines 413 and 422) (BASF 2004). Groups of ten male and ten female Wistar rats were exposed to vapours of tert-butyl acrylate for 6 hours/day and 5 days/week to concentrations of 20, 60 and 180 ppm (corresponding to 0.106, 0.319 and 0.956 mg/L). A concurrent control group was exposed to clean air. The males were treated for approx. 13 weeks (10 weeks premating, 3 weeks mating and post mating). In females treatment lasted from 10 weeks premating, during mating and gestation through day 4 after delivery (approx. 15 weeks). No effects were seen in the low (20 ppm) and mid (60 ppm) dose group.

The number of liveborn and stillborn pups, the live birth index amounted to 100% in test groups exposed to 0, 20 and 60 ppm and 80% in the high dose group (180 ppm). In the high dose group also significantly impaired post-delivery development and viability of the offspring was noted reducing the viability index to 20 % (97 % in control). The average pup body weights and body weight gains were significantly lowered in the high dose group. Pup clinical observations did not show clinical signs of toxicity, no malformations were described. Based on these data, the NOAEC for fetotoxicity was determined to be 0.319 mg/L (60 ppm) and for teratogenicity the NOAEC was ≥ 0.956 mg/L (180 ppm).

 

Additionally, data regarding developmental toxicity are available for the structural analogue n-Butyl acrylate:

 

For n-butyl acrylate a developmental toxicity study according to OECD414 and GLP was performed (BASF 2017). 25 inseminated New Zealand White rabbits were exposed orally (gavage) to 50, 150 and 400 mg/kg bw/day during gestation days 7 through 28.All females in the control, 50, 150, and 400 mg/kg/day groups survived to the scheduled necropsy. No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. A temporary substance-related absence of a mean body weight gain (0 g) was noted in the 400 mg/kg/day group on the first day of dose administration (gestation day 7-8) and resulted in a slightly lower mean body weight gain in this group compared to the control group during gestation days 7-10. Mean body weight gains in the 400 mg/kg/day group were similar to the control group for the remainder of the treatment period. and therefore were considered non-adverse. There were no substance-related macroscopic findings noted at the scheduled necropsy on gestation day 29 in the 50, 150, and 400 mg/kg/day groups. In addition, mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Intrauterine growth and survival in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. In addition, no test substance-related external, visceral, and skeletal malformations or developmental variations were noted in the offspring at any dosage level. Therefore, a NOAEL of 400 mg/kg bw is derived for both maternal and developmental toxicity based onthe absence of adverse effects.

For n-butyl acrylate an extended one generation study according to OECD 443 and GLP was performed. 30 Crl:CD(SD) rats were exposed to 20, 50 and 150 mg/kg bw/day by oral (gavage) exposure route (BASF, 2017). With some exceptions, the analyzed dosing formulations were within the acceptable range for suspensions (85% to 115%) and were homogeneous. Analyzed concentrations above the acceptance criteria had no impact on the study. The vehicle and test substance formulations were administered to the F0 males and females for a minimum of 70 consecutive days prior to mating. The F0 females continued to be dosed throughout mating, gestation, and lactation, through the day prior to euthanasia, for a total of 132-136 doses. Test substance-related histologic changes were observed in all dosage groups in the F0 generation. Epithelial hyperplasia and/or hyperkeratosis was observed in the non-glandular stomach in all test substance-treated groups examined. Although associated with the gross observation of thickened non-glandular stomach, the microscopic changes were not associated with any clinical pathology, organ, or body weight changes and were considered non-adverse. Additional non-adverse findings were noted in the high dose F0 group (biliary hyperplasia and mineralization at the corticomedullary junction). Mean body weights, body weight gains, food consumption, and food efficiency of females were unaffected by test substance administration. There were no test substance-related effects on the number of F1 pups born, live litter size, percentage of males at birth, F1 postnatal survival, clinical observations, anogenital distance, offspring body weights, necropsy findings, or developmental landmarks (areolae/nipple retention, vaginal patency, and balanopreputial separation). No test substance-related effects on clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were noted for F0 and F1 animals at any dosage level. In addition, no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroidstimulating hormone) were noted in F0 or F1 pups (on PND 4 and 21). Based on the absence of systemic toxicity in F0 females, a dosage level of 150 mg/kg day (highest dose tested)was considered to be the NOAEL. Based on the lack of effects for F1 litters, a dosage level of 150 mg/kg/day was considered to be the NOAEL for neonatal toxicity.  

Sprague Dawley rats were exposed to n-butyl acrylate vapour concentrations of 25, 135 and 250 ppm (corresponding to approx. 0.13; 0.71; 1.31 mg/L) for 6 hours per day on days 6 to 15 of gestations (BASF, 1979). Inhalation of 135 and 250 ppm of the test substance caused a significant reduction in maternal body weight gain, as well as irritation to the nose and eyes. At the end of the exposure period, the weight gain was comparable to the controls. The two highest exposure concentrations caused embryo lethality, as evidenced by a dose-dependent increase in post-implantation loss. The 25-ppm dose did not lead to any signs of maternal toxicity or embryo lethality. No signs of organ changes or skeletal abnormalities were observed in the fetuses at any concentration. The NOAEC for maternal and developmental toxicity was 25 ppm (0.13 mg/L), and the NOAEC for teratogenicity was 250 ppm (1.31 mg/L).

In a study where the developmental toxicity of seven acrylates was investigated, groups of 25 pregnant rats were exposed to 0, 100, 200 and 300 ppm n-butyl acrylate (corresponding to approx.0.52, 1.05, and 1.57 mg/L) for 6 hrs/day from days 6 through 20 of gestation (Saillenfait 1999). A NOAEC for maternal toxicity could not be determined since the absolute weight gain was significantly reduced in all dose groups in a concentration-related manner. No treatment-related effects were reported in terms of numbers of implantation sites, live fetuses, non-live implants or resorptions. Fetal body weight was significantly reduced at 200 and at 300 ppm. A few sporadic malformations were seen in the 300 ppm and the control group. There was no evidence of treatment-related effects on the incidence of external and visceral variations. The incidence of individual skeletal variations (mainly incomplete ossification of sternebrae and of vertebral centra) was similar in the control and treated groups. No NOAEC could be derived for maternal toxicity. The LOAEC for maternal toxicity was 100 ppm (0.52 mg/L), the NOAEC for developmental effects (fetotoxicity) was 100 ppm (0.52 mg/L), and the NOAEC for developmental effects (teratogenicity) was the highest concentration tested of 300 ppm (1.57 mg/L).

 

In a gavage study, pregnant CD-1 mice were administered n-butyl acrylate dissolved in cottonseed oil (Rohm and Haas, 1979). Concentrations of 0, 100, 1000, 1500, 2000, 2500, 3000 and 4000 mg n-butyl acrylate/kg bw were administered from gestation day 6 to 15. No animals survived in the high dose group. At 3000 and 2500 mg/kg bw 2 of 30 animals died; at 2000 mg/kg bw 1 of 29 died; at 1500 mg/kg bw 1 of 27 died; and at 1000 mg/kg bw 1 of 30 died. At the 1500 mg/kg bw dose and higher, average maternal body weight gain was significantly reduced. Fetal body weights were significantly reduced at doses of 1500 mg/kg bw and above. At 2500 and 3000 mg/kg bw, the percentage of resorptions was significantly increased. At 100, 1000, 1500 and 2000 mg/kg bw, and in the control group, variations and malformations occurred sporadically on different sides (i.e. single cases of cleft palate, fused ribs, fused sternebrae, fused arches, extra arches, branched ribs) in a non-dose-dependent manner, with a slight dose-dependent increase when taking the sum of all events per dose group together. In the 2500 mg/kg bw and 3000 mg/kg bw groups, the number of fetuses with external and skeletal malformations and variations (cleft palate, exencephaly, open eyes, fused arches, fused ribs) was significantly increased. Taking maternal mortality and reduced weight gain into account; the NOAEL for maternal toxicity was 100 mg/kg bw. The NOAEL for developmental toxicity was 1000 mg/kg bw and the NOAEL for teratogenicity was 2000 mg/kg bw. The observed effects on the offspring are most likely the result of excessive maternal toxicity and therefore a secondary effect.

 

Additionally, data regarding developmental toxicity are available for the structuralanalogue n-Methyl acrylate:

A prenatal developmental toxicity study in rabbits as second species was conducted with methyl acrylate according to OECD TG 414 for the Acrylate Task Force (BAMM 2009). 25 inseminated female Himalayan rabbits per group were whole-body exposed for 6 hrs/day, 5 days/week over a time period of 23 consecutive days (gestation days (GD) 6–28) to methyl acrylate vapours at target concentrations of 0, 5, 15, and 45 ppm. Analytical concentrations of 4.9, 15.7, 44.2 ppm (corresponding to approx. 0.0174, 0.0553, 0.1556 mg/L) were measured. On gestation day 29 the does were sacrificed and submitted to gross and histopathological examination (nasal cavities, larynx, trachea, lungs, mediastinal lymph nodes, all gross lesions). Examinations of ovaries and uterine content of the does included: determination of the weight of the unopened uterus, of the number of corpora lutea, of the number and distribution of implantation sites, and calculations of conception rate and pre- and post-implantation losses. Fetal examinations were performed on all fetuses per litter (external, soft tissue, skeletal) except head examinations that were done on half of the fetuses per litter.

There were no test substance-related effects on the does concerning food consumption, gross/net body weight, gestational parameters, uterine, placental and lung weights, as well as necropsy observations up to and including a dose of 45 ppm. The test substance caused a severe degeneration and atrophy of the olfactory epithelium at at least one focal area in the nasal cavity (distal levels III and/or IV) at the high-dose level (45 ppm). Though being local effects, such massive findings in the respiratory tract are likely to cause a considerable amount of distress in the affected maternal animals. Since distress is supposed to influence maternal homeostasis, this is considered to be a significant adverse effect on the maternal organism. The NOAEC for maternal toxicity was 15 ppm (0.0553 mg/L), or 0.0823 mg/L after correction of molecular weight.

 Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. Methyl Acrylate (MA) had no adverse effect on prenatal development of offspring at any of the dose levels tested (5, 15 and 45 ppm). Thus, the NOAEC for developmental effects (fetotoxicity) and the NOAEC for developmental effects (teratogenicity) was the highest concentration tested of 45 ppm (0.1556 mg/L). After correction for molecular weight, the NOAEC is 0.23 mg/L for the respective endpoints.

 

Additionally, data regarding developmental toxicity are available for the structural analogue ethyl acrylate:

 

The developmental toxicity of ethyl acrylate was evaluated in Sprague-Dawley rats after inhalation exposure for 6 hours/day, on gestation days 6 to 20. The exposure concentrations were 0, 25, 50, 100, or 200 ppm (corresponding to 0, 0.10, 0.21, 0.41, or 0.82 mg/L). Dose groups consisted of 17 to 19 pregnant rats. No maternal deaths were observed during the study.

Significant decreases in maternal body weight throughout exposure to 200 ppm were observed. There was no significant difference in the numbers of implantation sites and live fetuses, in the incidence of non-live implants and resorptions, or in the fetal sex ratio. Fetal body weights were significantly reduced at 200 ppm (7-8 % lower than control). No treatment-related increase in embryo/fetal lethality or fetal malformations was observed at any dose level. The incidence of external, visceral, and skeletal variations was similar to controls. The NOAEC for maternal and fetal toxicity was 100 ppm(0.41 mg/L). The NOAEC for developmental toxicity and teratogenicity was 200 ppm (0.82 mg/L), the highest exposure concentration tested.(Saillenfait et al., 1999). After correction for molecular weight, the NOAEC is 0.53 mg/L and 1.05 mg/L for maternal and fetal toxicity, and developmental toxicity respectively.

 Additionally, data regarding developmental toxicity are available for the structuralanalogue ethyl acrylate(see read-across justification):

Pregnant Sprague-Dawley rats were exposed to 0, 50, or 150 ppm of ethyl acrylate (corresponding to 0, 0.21, 0.62 mg/L) for 6 hours/day during days 6 through 15 of gestation (the period of major organogenesis) (IATG, 1980). Maternal toxicity as evidenced by decreased body weight gain, decreased food consumption and increased water consumption was noted among rats exposed to 150 ppm of ethyl acrylate. No maternal toxicity was evident in pregnant females exposed to ethyl acrylate at the 50 ppm level. External and internal examination of pups did not reveal a statistically significant increase in any major malformation, but a low incidence of malformed fetuses was observed at the 150 ppm level of ethyl acrylate (not statistically significant). Based on these data, inhalation of ethyl acrylate vapours by rats at a concentration of 50 or 150 ppm during major organogenesis was not considered to be teratogenic. In the presence of maternal toxicity at 150 ppm, a slight increase (not statistically significant) in malformed fetuses was observed. At 50 ppm, there was neither maternal toxicity nor an adverse effect on the developing embryo and fetus of rats. Thus, the NOAEC for maternal toxicity was 0.21 mg/L, and for developmental toxicity and teratogenicity 0.62 mg/L in this study, again the highest exposure concentration tested (IATG 1982). After correction for molecular weight the NOAEC is 0.27 mg/L for maternal toxicity and 0.79 mg/ L for developmental toxicity and teratogenicity.

 

Additionally, data regarding developmental toxicity are available for the structural analogue2-Ethylhexyl acrylate:

2-EHA was investigated during a study on the relative developmental toxicities of a set of various acrylates (acrylic acid, methyl acrylate, butyl acrylate, hydroxyethyl acrylate, hydroxypropyl acrylate) in Sprague-Dawley rats (Saillenfait et al. 1999). For the investigation with 2-EHA groups of 23 to 25 dams were exposed (6 hours/day, whole-body) to atmospheres containing 2-ethylhexyl acrylate (99.7 % purity) at 0, 50, 75, and 100 ppm (approximately 0.38, 0.56, and 0.75 mg/L) during day 6 to day 20 of gestation. Maternal food consumption was measured for the intervals of g.d. 6 -13 and of g.d. 13-21. Maternal body weights were recorded on g.d . 0, 6, 13, and 2l. Dams were sacrificed on day 21 of gestation and the uteri were removed and weighed. The number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri, which had no visible implantation sites, were stained with ammonium sulfite (10 %) for the detection of early resorptions. At sacrifice live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of live fetuses from each litter were examined for either internal soft tissue or for skeletal changes. There were no maternal deaths in any of the treatment groups. Dams from the 100-ppm groups showed an absolute weight gain of 24±16 g through the period of exposure, which was lower and statistically significantly different from that of the concurrent control group (42 ± 11 g). Also food intake of 24 ± 3 g food/dam/day through the period of exposure of the 100-ppm group was somewhat lower and statistically significantly different in comparison to that of the concurrent control group (27 ± 2 g food/dam/day). No adverse effects were observed on the mean number of implantation sites per litter and on the mean number of live fetuses per litter in any of the 2-EHA exposed groups. The incidences of non-live implants (3.7 - 6.4 %) and of resorption sites per litter (3.7 - 6.1 %) in the treated groups were lower than those of the concurrent control (both 10.1 %). This observation, however, is not considered to be of toxicological significance. Mean fetal body weights were slightly lower in the treated groups, however not statistically significantly different from that of the concurrent control fetuses. Sex ratio was unaffected. No significant differences were observed between the control and the 2-EHA-treated groups in the incidences of gross anomalies or of visceral or skeletal malformations or variations.

 

In summary, no embryotoxic, teratogenic or fetotoxic properties of 2-EHA had been revealed from this study for concentrations of up to and including 100 ppm. Due to technical limitations exposure to higher concentrations could not be tested. Based on slightly reduced food intake and lower maternal weight gain at the higher exposure level a NOAEC for maternal toxicity of 75 ppm (approximately 0.56 mg/L) was derived from this study. No embryo-/fetotoxic effects were revealed even at the highest tested concentration at which some signs of maternal toxicity had been observed. Therefore, a NOAEC for developmental toxicity of 100 ppm (approximately 0.75 mg/L) was derived from this study. After correction for molecular weight, the NOAEC is 0.4 mg/L for maternal toxicity and 0.52 mg/L for developmental toxicity.

 

 

Conclusion

The overall evidence based on the data on tert-butyl acrylate itself as well as from the read-across substances methyl acrylate, ethyl acrylate, n-butyl acrylate and 2-ethylhexyl acrylate gives no indications that this class of chemicals has a teratogenic potential. Only in overt maternal toxic concentrations / doses fetotoxic effects, probably as secondary effect were described for this class of acrylates. In analogy to nBA the DNEL for tBA is based on the OEL of nBA of 2 ppm. Using the DNEL derivation according to the ECHA Guidance on information requriments and chemical safety assessment, Chapter R.8: Characterisation of dose (concentration)-response for human health supports the OEL also for tBA .

The German MAK Commission and the EU Scientific Committee on Occupational Exposure Limits (SCOEL) derived the OEL of 2 ppm which was taken as DNEL based on the most sensitive endpoint the local irritation of the olfactory epithelium of the nasal mucous membranes which occurred in the 2 year inhalation study at 15 ppm. Based on the above mentioned studies, not indicating any suspicion for a teratogenic effect in much higher concentrations, further animal studies will not provide more safety to workers.

Justification for classification or non-classification

Based on the available data classification for reproductive/developmental toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 and GHS classification (GHS UN rev.7, 2017).