1-Butanamine, N-butyl-, reaction products with polyethylene-polypropylene glycol ether with trimethylolpropane (3:1) acrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Study period:

2012/2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 1996

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, adopted July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany, male and female rats were derived from different litters to rule out sibling mating
- Age at study initiation: (P) 10-11 wks;
- Weight at study initiation: (P) Males: 294 g; Females: 197 g, on average
- Fasting period before study: no
- Housing: individually (except during overnight mating and lactation) in Makrolon type M III cages
- Diet (e.g. ad libitum): Kliba maintenance diet mouse-rat “GLP” meal, ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr):15

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The test substance was applied as suspension, which was prepared at least once a week. An appropriate amount of the test substance was weighed, and corn oil was added up to the desired volume. The suspension was kept homogenous during administration by stirring with a magnetic stirrer.

The administration volume was 4ml/kg b.w.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance is poorly soluble in water.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 14 days, or until pregnancy was proven
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): single

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in corn oil for a period of 7 days at room temperature was proven before the start of the study (BASF Project No. 11L00386).
For homogeneity and concentration control analyses, each 6 samples of all concentrations was drawn at the start of the study and towards the end of the administration period. Homogeneity was confirmed, all concentrations measured were within 90-110% of the target concentration.

Duration of treatment / exposure:

males: 36 days
females: 51 days

Frequency of treatment:

daily, except to animals being in labor

Remarks:

Doses / Concentrations:
100, 300, 1000mg/kg (high dose was reduced to 600mg/kg on day 19)
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of a range-finding study - no severe signs of toxicity were observed in animals treated with 1000mg/kg of the test substance for 14 days.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on workdays, daily on weekends and public holidays
- Cage side observations: check for moribund animals, pertinent behavioral changes, signs of overt toxicity, parturation, littering and lactation behavior of the dams

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first administration, weekly thereafter
- The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, weekly thereafter with the following exceptions for females: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, except during mating

WATER CONSUMPTION: Monitored by daily visual inspection

OTHER (for details see entry for this study in the repeated dose section):
- Functional observation battery and motor activity measurement of 5 males and females per group 2 days prior to sacrifice
- Urinanalysis in 5 males and females per group one day prior to necropsy
- Clinicochemical and hematological examinations 5 males and females on the day of necropsy after fasting for 16h

Sperm parameters (parental animals):

Parameters examined in male parental animals:
testis weight, epididymis weight, stages of spermatogenesis in the male gonads

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical symptoms, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, externally and organs were assessed macroscopically

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 37
- Maternal animals: All surviving animals on day 52

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were weighed
- in all animals: epididymides, testes
- in 5 males and females of each group: adrenal glands, brain, heart, kidneys, liver, spleen, thymus
The uteri of all cohabited female parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations.
The following tissues were examined histotechnically in at least 5 animals per sex of the control and high dose group unless noted otherwise
adrenal glands, gross lesions (all affected animals in all dosage groups), bone marrow (femur), brain, cecum, cervix, coagulation glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (auxillary and mesenteric), ovaries, oviducts, prostate gland, peyer's patches, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, forestomach (all animals from all dose groups), glandular stomach, testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

Postmortem examinations (offspring):

NECROPSY
- All surviving pubs were subjected to postmortem examinations: external examination and macroscopic examination of organs.

Reproductive indices:

For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
Male mating index (%) = number of males with confirmed mating (vaginal sperm detected in females) / number of males placed with females x100
Male fertility index (%) = number of males proving their fertility (implants in utero) / number of males placed with females x100

For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
Female mating index (%) = number of females mated (vaginal sperm detected) / number of females placed with males x100
Female fertility index (%) = number of females pregnant (implants in utero) / number of females mated (vaginal sperm or implants in utero) x100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x100

Offspring viability indices:

Live birth index (%) = number of liveborn pups at birth / total number of pups born x100
Viability index (%) = number of live pups on day 4 after birth / number of live pups on the day of birth x100

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In test group 3 (1000 and 600 mg/kg bw/d) female animal No. 80 was found dead on study day 17 (Mating Day 3), female animal No. 74 was sacrificed moribund on study day 19 (GD 2), and male animal No. 32 was found dead on study day 35. These premature deaths were assessed as being related to the test substance. One female animal of test group 2 (300 mg/kg bw/d) was also found dead on study day 44. The animal died because of a gavage error. Therefore, a relation to treatment was excluded.

Almost all animals of both sexes of the mid and high dose group showed mostly slight, sometimes moderate salivation within 2 hours after the test substance administration on several days of the study. The same was true for individual low dose animals. From the temporary, short appearance immediately after dosing it could be assumed that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse.

Animal No. 74 of test group 3 (1000 and 600 mg/kg bw/d) showed poor general condition, piloerection and respiration sounds on GD 1 and GD 2. Therefore, the animal was sacrificed in a moribund condition on GD 19. Animal No. 75 of the same group showed semiclosed eyelids, poor general condition, hunched posture, smeared fur, piloerection (up to GD 7) and gasping on GD 2 and respiration sounds on GD 3. The same animal showed respiration gasping and sounds on LD 1. These findings were assessed as being related to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No treatment-related changes in body weight data were observed in animals of all test groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
One low dose female showed bloody red vaginal discharge and was unable to deliver on GD 22 and 23 as well as LD 0. The finding was assessed as being incidental as no dose-response relationship occurred.
For all except one high dose male (and corresponding female) mating was confirmed. Of these, 2 control males, one low dose did not generate pubs. 2 high dose males also failed to produce offspring due to premature death of the corresponding female. The gestation index varied between 100% (control), 88.9% (low dose), 100% (mid dose), 87.5% (high dose). The rate of liveborn pubs was also unaffected by the test substance as stillborn pubs only occurred in the control and high dose group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute thymus weights were significantly decreased in high dose male animals when compared to control group animals. This finding was not considered to be treatment related, since no relative organ weights were significantly altered.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Erosions, ulcers, and in one case a thickened forstomach were observed in all high dose male and females (with the exception of one female) and 5 mid dose females and 4 mid dose males. In the animals that did not become pregnant, no gross lesions of reproductive organs were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Five male and 6 female animals of test group 3 (1000 and 600 mg/kg bw/d) as well as each 2 males and females of test group 2 (300 mg/kg bw/d) revealed multifocal erosions/ulcers in the forestomach. Accompanying these erosions/ulcers a mild to extreme diffuse or focal squamous hyperplasia with hyperkeratosis and a mild to severe submucosal edema was present. When the hyperplasia of the squamous epithelium was focal, it was located at the cardia region (stomach entrance). Within the edema a higher number of inflammatory cells were observed. Not all animals with a macroscopically diagnosed erosion/ulcer showed this finding. But the consistency of the accompanying findings (hyperplasia with hyperkeratosis and edema) which were observed in animals with and without erosion/ulcer led to the conclusion that this all belongs to the same complex. As the erosions/ulcer that could be seen microscopically were very small it is possible that they were not on the level of section. But taken all these findings together they point towards an irritating effect of the substance on the squamous epithelium of the forestomach. Especially the cardia region (entrance of the stomach) was affected.
The three animals of test group 3 (1000 and 600 mg/kg bw/d) which were found dead or were sacrificed in a moribund (animal Nos. 32, 74 and 80) did not show any other findings that could explain the death beside the ones in the stomach. Two of them revealed a moderate to severe thymus atrophy which was regarded to be a consequence to the stress caused by the findings in the forestomach. But it is not clear whether these observed findings were the cause of the death although death was regarded to be a consequence to the treatment with the test substance.
All investigated nonpregnant females as well as their male mating partners did not show relevant histopathological findings that could explain infertility.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 300 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: death and poor general condition in high dose animals

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

>= 100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: irritation in the stomach

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

>= 600 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no effect

VIABILITY (OFFSPRING)
Stillborn pubs were only observed in the control (1 pub) and low dose group (2 pubs), which were assessed as incidental. One low dose pub was cannibalized, all other pubs from all dose groups survived until scheduled necropsy on PND4.

No difference in sex ratio was observed.

CLINICAL SIGNS (OFFSPRING)
No adverse clinical signs were noted.

BODY WEIGHT (OFFSPRING)
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values.

GROSS PATHOLOGY (OFFSPRING)
A small liver with a discolored liver lobe (pale) was observed in 1 male pup of test group 1 (100 mg/kg bw/d). These findings were assessed as being spontaneous in nature and without biological relevance.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

>= 600 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no effect

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no data available.

The test item is Propylidynetrimethanol, ethoxylated, propoxylated, esters with acrylicacid, reaction products with 1-butanamine, n-butyl (Cas:173011-06-8). The concentration of EO/PO is >1 <6.5. The chosen read-across substances is Propylidynetrimethanol, ethoxylated, esters with acrylicacid, reaction products with 1-butanamine, n-butyl (Cas: 195008-76-5) concentration of. EO>1 <6.5.

In general basic physicochemical properties such as water solubility, vapour pressure and the partition coefficient (LogPow) are quite similar. The water solubility was 0.9 g/l the vapour pressure 2.3 hPa and the logPow 4.2 and 0.8 g/l, 1.6 hPa and the logPow 3.5 for the read-across substance.

Both molecules show no acute toxicity after oral or dermal exposure and did not induce gene mutations in bacteria. Both showed no skin irritation and Cas: 195008-76-5 was slightly eye irritating. Due to the very similar structure, physicochemical properties and almost identical results in the toxicological studies mentioned above, comparable results are also expected for the endpoints for which read across was performed.

Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- was administered daily in corn oil via gavage to groups of 10 male and 10 female Wistar rats at dose levels of 100, 300, and 1000mg/kg. Because of the premature death of 2 female animals, the high dose was reduced from 1000 to 600 mg/kg bw/d from study day 19 onwards. The duration of treatment covered 36 days in males and 51 days in females, including premating, mating, gestation and 4 days of lactation.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and at weekly intervals thereafter. Food consumption of the F0 parents was determined once weekly during premating. For the dams food consumption was determined for gestation days 0-7, 7-14, 14-20 and lactation days 1-4. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 0) and on PND 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. Clinicochemical and hematological examinations as well as urinalyses were also performed towards the end of the administration period in 5 animals per sex and test group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Of the high dose animals, one male and one female were found dead, and one female was sacrificed in moribund condition. Piloerection, smeared fur, respiratory sounds, poor general state, hunched posture, and semiclosed eyelids were observed in single animals of this group at different time points. Pathology revealed erosion/ulcer in the forestomach of all male and 9/10 female high dose animals, mild to severe diffuse or focal hyperplasia with hyperkeratosis in the forestomach of all animals, and mild to severe submucosal edema in the forestomach of 8 male and 3 female animals. Animals receiving 300mg/kg b.w. showed comparable signs of local irritation in the forestomach, though the incidence was clearly reduced. No clinical signs were noted in this group and in low dose animals, which also showed no local irritation in the stomach. Reproductive performance was unaffected in all groups. No deviations from control animals were observed in the pubs from all groups. Thus the NOAEL for fertility and developmental toxicity was set to 600mg/kg b.w. Though the clinical signs and deaths of high dose animals are most likely secondary to the severe irritation in the stomach, systemic toxicity cannot be completely excluded. Following the precautionary principle, the NOAEL for systemic effects was set to 300mg/kg b.w., and the NOAEL for local irritation was set to 100mg/kg.

Short description of key information:
combined rep. dose / dev. screening study, rat (BASF 2013, GLP, OECD422): no effects on fertility nor developmental defects observed.

Effects on developmental toxicity

Description of key information

combined rep. dose / dev. screening study, rat (BASF 2013, GLP, OECD422): NOAEL (dev.) = 600mg/kg (highest dose)
read across substances:
Dibutylamine, hydrochlorid salt (6287-40-7): teratogenicity, rat (BASF 2010, GLP, OECD 414): NOAEL (dev.) = 150mg/kg (highest dose)
TMPeoTA (28961-43-5): teratogenicity, rat (Cytec 1984, GLP, OECD 414): NOAEL (dev.) = 1000mg/kg (highest dose)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

As already discussed for effects on fertility, Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- was tested in an OECD 422 study, in which viability and macroscopic abnormalities of the pubs are also assessed. No difference to control pubs were observed in this study. Though this study does not fulfill all requirements to completely exclude developmental defects, it supports the negative results obtained with the two components of the test substance: Propylidynetrimethanol, ethoxylated, esters with acrylic acid (TMPeoTA, CAS 28961 -43 -5) and 1-Butanamine, N-butyl- (tested as the hydrochloric salt, CAS 6287 -40 -7). For all three substances, the primary effect in maternal animals is local irritation in the stomach. Most likely, all further systemic effects like weight loss and poor general state are secondary to the irritant properties. No specific target organ toxicity was observed in all three cases.

TMPeoTA and Dibutylamine-HCl were tested according to (or similar to ) the OECD 414 guideline for prenatal developmental toxicity. 30 Spraque Dawley female rats were treated orally with 1000mg/kg TMPeoTA in corn oil from gestation days 6 -15. The animals were sacrificed on gestation day 20, and uteri and ovaries were examined for corpora lutea, implantations, early resorptions, and late resorptions. All fetuses were examined externally. Sex ratio and viability were determined. Half of the fetuses were subjected to soft tissue examination, while the other half was examined for sceletal abnormalities. A significant decrease in body weight of the dams was observed from gestation days 6 -16. Clinical signs included salivation, urogenital matting and hair loss from various body surfaces. No embryotoxicity or teratogenicity was noted.

CAS 6287 -40 -7 was administered by gavage to 25 pregnant female Wistar rats at concentrations of 15, 50, and 150 mg/kg in water from gestation days 6 -19. The animals were sacrificed on gestation day 20, and the gravid uterus weight, number of corpora lutea, number of implantation sites, early resorptions, late resorptions, the number of dead fetuses, and the sex of all fetuses were determined. All fetuses were examined externally, and subjected to soft tissue and sceletal examinations. There were no test substance-related effects on the dams concerning mortality, food consumption, gross and corrected (net) body weights, gestational parameters, uterine and placental weights, as well as necropsy observations up to and including a dose of 150 mg/kg bw/day. Some mid- and high-dose dams showed transient salivation for a few minutes immediately after each treatment, which was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. Salivation was not observed after treatment with the low doses. Body weight gain of the high-dose rats was significantly reduced (by 63%) at initiation of treatment (GD 6-8) but recovered afterwards. The increase of the red blood cell counts, the hemoglobin as well as the hematocrit values in the dams on GD 20 beginning in the 50 mg/kg bw/d dose group was an indication of hemo-concentration, most probably because of stress. This assumption was confirmed by the increase of the total white blood cell counts, which was due to higher lymphocyte counts, in the rats of the 150 mg/kg bw/d dose group. Correspondingly, the increase of the urea as well as of the cholesterol values in the dams beginning in the 50 mg/kg bw/d dose group can be interpreted as a consequence of stress accompanied by an increased protein metabolism. Additionally, the latter effect can be partly explained by an increased bone metabolism, which was also expressed by increased inorganic phosphate and calcium blood levels in the rats beginning in the 50 mg/kg bw/d dose group. A slight affection of the liver cells in the dams of the 150 mg/kg bw/d dose group was indicated by the increase of the ALT activity. Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses, fetal body weights, and on fetal morphological structures. 

Justification for classification or non-classification

No effects on fertility were observed in an OECD 422 study using Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with 1-Butanamine, N-butyl-. In this study, also no difference in pubs from control and treated animals were noted. In addition, one study each for prenatal developmental toxicity exists for the two components of the reaction mass, CAS 28961 -43 -5 and 6287 -40 -7. No treatment related effects on embryotoxicity or fetal malformations were found in these studies. Considering the results from all three studies, Propylidynetrimethanol, ethoxylated, propoxylated esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- is not expected to cause developmental toxicity, teratogenicity or affect fertility. Therefore, it does not require classification according to 67/548/EEC or CLP/EU-GHS concerning reproductive toxicity.