3-butoxypropylamine

Endpoint summary

Description of key information

Repeated dose toxicity - oral: A key K1 study in male and female Wistar rats in a combined repeated dose toxicity test with reproductive/developmental screening was conducted according to OECD guideline 422 (Edwards, 2018). The NOAEL is established to be 90 mg/kg body weight/day.

Repeated dose toxicity - dermal or inhalation: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation or dermal route of exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-29 to 2019-11-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: (EC) No 440/2008

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name: BOPA, 3-Butoxypropylamine
- CAS number: 16499-88-0
- Physical state/appearance: clear, colorless liquid
- Batch number: PFW160573
- Purity: 99.39%
- Expiry date: 2018-09-26

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Ltd., Oxon, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Males: approx. 11 weeks old at the start of the treatment
Females: approx. 12 weeks old at the start of the treatment
- Weight at study initiation:
Males: 292-350 g
Females: 193-231g
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male:one female basis within each dose group.Following evidence of successful mating, the males were returned to their original cages and mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum, pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.)
- Water (e.g. ad libitum): ad libitum, mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY:
The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hours) : at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE : From 2017-12-19 to 2018-02-20

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe.
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- the test item was prepared at the appropriate concentrations as a solution in arachis oil BP.
- the formulations were shown to be stable for at least 4 hours. Formulations were therefore pepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration in vehicle: 0, 7.5, 15, 30/22.5* mg/mL (*dose level and concentration reduced from day 11)
- Amount of vehicle (if gavage): 4 mL/kg of Arachis oil BP. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
- Lot/batch no. (if required): Not specified
- Purity: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The analytical procedure was successfully validated for the test substance in Arachis Oil with reqspect to the specificity of chromatographic analysis, the linearity of detector response, method accuracy and precision.
- The homogeneity and stability of the test substance in Arachis Oil formulations was assessed with respect to the level of concentration at nominal concentrations of 2.5 mg/mL and 50 mg/mL.
- Samples of test item formulations were taken on two occasions and analyzed for concentration of the test item.

Duration of treatment / exposure:

- Males: approx. 6 weeks
- Females: up to 8 weeks (including a two-week pre-pairing phase, pairing, gestation and early lactation)

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control)

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Group 2 (low dose group)

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

Group 3 (Intermediate dose group)

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

Group 4 (high dose group), up to day 11

Dose / conc.:

90 mg/kg bw/day (nominal)

Remarks:

Group 4 (high dose group), from day 11 until the end fo the study

No. of animals per sex per dose:

48 males and 49 females in total; 12 males and 12 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (including a 14-day repeated dose oral (gavage) range-finding toxicity study in the rat (Covance Study Number CM26GG)
- Treatment volume : 4mL/kg body weight of Arachis oil BP.
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable)
- Parameters: overt signs of toxicity, ill-health, behavioral change

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- During the pre-pairing period : weekly food consumption was recorded for each cage of adults.
- For males after the mating phase: weekly food consumption was recorded for each cage of adults.
- For females showing evidence of mating: food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20.
- For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14.

FOOD EFFICIENCY: Yes
- For males: was calculated retrospectively throughout the study period (with the exception of the mating phase).
- For females during the pre-pairing phase: was calculated throughout retrospectively the study period (with the exception of the mating phase).
- Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Water intake was observed daily by visual inspection of water bottles for any overt changes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination (Day 43 for males and Day 13 post partum for females)
- Anaesthetic used for blood collection: No. Blood samples were obtained from the lateral vein.
- Animals fasted: No
- Number of animals per group: five males and five females selected from each test and control group
- Parameters checked: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic)
- Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination (Day 43 for males and Day 13 post partum for females)
- Animals fasted: No
- How many animals: five males and five females selected from each test and control group
- Parameters checked : Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids, Calcium (Ca++).

OTHER:
FUNCTIONAL OBSERVATIONS
- prior to start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on days 4, 11 and 18 post coitum and for littering females on days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIORAL ASSESSMENT
- detailed individual clinical observations for each animal using a purpose built arena
- parameters: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation

FUNCTIONAL PERFORMANCE
- Motor activity:
Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period.
- Forelimb/Hindlimb Grip Strength: An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made.

SENSOR REACTIVITY
- Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
- Parameters observed : Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

THYROID HORMONE ANALYSIS
- serum and plasma samples were taken from all adult males and females at termination.

Sacrifice and pathology:

SACRIFICE
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45.
Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.

GROSS NECROPSY
- Post mortem procedures were performed on all adult animals and included any animal found dead or killed in extremis during the study.
- a full external and internal examination

ORGAN WEIGHTS
- For five males and five females selected from each test and control group
The following organs dissected free from fat and weighed before fixation : Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary (weihed partially fixed), Prostate, Seminal vesicles (with coagulation gland), spleen, Testes, Thymus, Thyroid (weighed partially fixed with parathyroid), Uterus (weighed with cervix and oviducts)
- For all remaining animals, the following organs were weighed: Epididymides, Prostate, Seminal Vesicles (with coagulation gland), Ovaries, Pituitary (weighed after partial fixation), Thyroid (weighed after partial fixation with parathyroid), Uterus (weighed with Cervix).

HISTOPATHOLOGY
- from five males and five females selected from each test and control group had the following organs preserved in buffered 10% formalin.
- The tissues were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination.
- Tissues / organs collected: Adrenals, Aorta (thoracic), Bone and bone marrow (femur including stifle joint), Bone and Bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Cowpers glands, Duodenum, Esophagus, Eyes (retained in Davidson's Fluid), Glans penis, Gross lesions, Heart, Ileum (including peyer's patches), Jejunum, Kidneys, LABC (levator ani-bulbocavernous) muscle, Liver, Lungs (with bronchi), Lymph nodes (mandibular and mesenteric), Mammary gland, Muscle, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles (with coagulation gland), Skin, Spinal cord (cervical, mid-thoracic and lumbar), Spleen, Stomach, Testes (retained in Modified Davidson's Fluid), Thymus, Thyroid/Parathyroid, Trachea, Urinary bladder, Uterus and Cervix (with oviducts), Vagina

Statistics:

The homogeneity of variance from mean values was analyzed using Bartlett’s test.
Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates.
Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data.
If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group.
Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing.
Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test.
Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the KruskalWallis test which if significant was followed by the Mann-Whitney "U" test.
Dose response relationships were also investigated by linear regression. Where the data were unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The majority of animals of either sex treated with 60 or 120/90 mg/kg bw/day displayed increased salivation for the majority of the treatment period; albeit at a lower extent at the lower dose level. Increased salivation was restricted to one male at 30 mg/kg bw/day on one occasion. Noisy respiration was seen in all animals of both sexes dosed with 120/90 mg/kg bw/day on a number of occasions throughout the study. At 60 mg/kg bw/day noisy respiration was seen on several occasions on four males and five females during the treatment period; in addition only one male and two females at 30 mg/kg bw/day showed the same observation. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and are considered not to represent an adverse effect of treatment.
Incidental observations include one male dosed at 120/90 mg/kg bw/day with staining around the snout on Day 29. One male dosed with 120/90 mg/kg bw/day showed pilo-erection and hunched posture on Day 24; one female dosed with 120/90 mg/kg bw/day showed piloerection on Day 1 after dosing. One female treated with 30 mg/kg bw/day exhibited staining around the eyes on Days 50 and 51. These observations are considered to be incidental and of no toxicological significance.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three females dosed with 120 mg/kg bw/day were killed in extremis.
- Female 94 (120 mg/kg bw/day) was killed in extremis on Day 2 due to adverse clinical signs including noisy, labored and gasping respiration, pilo-erection, lethargy, hunched posture and distended abdomen. Necropsy observations included gaseous distention of the cecum, colon, duodenum, ileum, jejunum, rectum and stomach. At histopathology there was marked necrosis in the trachea and death is considered to be related to this change, which possibly reflects esophageal reflux.
- Female 90 (120 mg/kg bw/day) was killed in extremis on Day 5. Previously the animal had shown instances of noisy respiration, and on Day 5 was observed to have noisy and gasping respiration, decreased respiratory rate, pilo-erection, lethargy and hunched posture. Necropsy findings included gaseous distension of the duodenum, ileum, jejunum and stomach. At histopathology there was marked necrosis in the trachea and death is considered to be related to this change, which possibly reflects esophageal reflux.
- Female 91 (120 mg/kg bw/day) was killed on Day 6. The animal displayed similar observations as female 90, with instances of noisy respiration on the day before it was humanely killed, and observations of noisy, labored and gasping respiration, decreased respiratory rate and pilo-erection on Day 6. Necropsy showed gaseous distension in the stomach, and a thin non-glandular region of the stomach, but no notable changes were apparent at histopathology.
- On Day 11, the high dose level was reduced from 120 to 90 mg/kg bw/day.
- One female dosed with 120/90 mg/kg bw/day was found dead during the bleeds procedure on Day 13 post partum (Day 51 of the study). Only occasional observations of noisy respiration and increased salivation were noted prior to the animal being found dead. No notable changes were apparent at necropsy or histopathology and this death was considered to be incidental and unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- For males dosed with 120/90 mg/kg bw/day: weight gains were statistically significantly lower (p<0.01 and p<0.05, respectively) than controls for the first two weeks of dosing. Following the reduction of the high dose level from 120 mg/kg bw/day to 90 mg/kg bw/day the subsequent body weight gains for these animals generally became comparable to controls (from Week 3).The overall body weight gain for males treated with 120/90 mg/kg bw/day was 19% lower than controls, which is considered to be due to the initial effects on body weight gains during the first two weeks of treatment.
- For males treated with 30 or 60 mg/kg bw/day: no effects were noted.
- All treated female dose groups: weight gains were generally comparable to controls during maturation, gestation and lactation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- For males dosed at 120/90 mg/kg bw/day: food consumptions for males were slightly lower than controls during the first two weeks of treatment. Improvement was noted for the remainder of the treatment period.
- For males dosed at 30 and 60 mg/kg bw/day: no effect on food consumption throughout the study.
- For females dosed at 120/90 mg/kg bw/day: slightly lower than controls during the first week of dosing (maturation)
- For females dosed at 30 and 60 mg/kg bw/day: food intake were similar to controls during maturation, the gestation and lactation periods.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

- For males dosed at 120/90 mg/kg bw/day: food conversion efficiency was slightly lower than controls during Weeks 1 and 2, which correlate to the body weight changes and food intake during this time.
- For females dosed at 120/90 mg/kg bw/day: no effects were noted.
- For males and females dosed at 30 or 60 mg/kg bw/day: no effects were noted.

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

no effects observed

Description (incidence and severity):

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of hematology parameters did not indicate any obvious effect of treatment for either sex at 30, 60 or 120/90 mg/kg bw/day.

- For males dosed at 120/90 mg/kg bw/day: platelet values were statistically significantly lower (p<0.05) than controls
- For males dosed at 30 or 60 mg/kg bw/day: mean corpuscular hemoglobin concentration values were statistically significantly higher (p<0.05) than controls.

For both paramaters above, 4/5 individual values were within normal background control ranges. In isolation, and in the absence of any supporting histopathological changes, these findings are likely to represent normal biological variation. They are considered to be incidental and of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 30, 60 or 120/90 mg/kg bw/day.
- Animals (either sex) treated at 120/90 mg/kg bw/day: showed statistical significance (p<0.05) in chloride levels compared to controls (males increased and females reduced). However, all individual values were within background control ranges. Hyperchloremia rarely occurs also and is usually accompanied as a part of parallel shifts in sodium levels. In the absence of any such findings, or any histopathological changes, this increase in males is considered to be a result of normal biological variation and of no toxicological significance. The result for female animals is also considered to be due to normal biological variation and of no toxicological significance.

- Males from all dose groups showed a reduction in alanine aminotransferase (ALAT) levels that attained statistical significance (p<0.05 - p<0.01) without a dose relationship. This enzyme is released into the bloodstream in excess of normal levels as a result of damage affecting the liver, thus a decrease in alanine aminotransferase is not an adverse effect. All values for treated males were within background control normal ranges, whereas 2/5 control values exceeded this range. Therefore, in absence of any histopathological liver findings, this finding considered likely to be due to unusually high control values, and considered to be of no toxicological significance.

- Females treated with 120/90 mg/kg bw/day : showed statistically significantly higher albumin/globulin ratios due to statistically significantly higher (p<0.01) levels of albumin. All values for females treated with 120/90 mg/kg bw/day were within normal historical control ranges, whereas 4/5 control values were within these ranges for both parameters. The remaining control value was significantly lower than the background control range for both albumin and albumin/globulin ratio, and this unusually low value is likely to be the cause of this statistical significance, rather than a true toxicological effect.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

-Behavioral assessments
There were no toxicologically significant effects detected in the behavioral assessments.
Seven males and four females exhibited noisy respiration during the first week of treatment while receiving 120 mg/kg bw/day. A further two females at 120/90 mg/kg bw/day showed noisy respiration during the first and third weeks of the gestation phase. These clinical signs were typical of post dose clinical observations seen and considered to be associated with the irritant nature of the test item rather than systemic or neurological effect.
One female treated with 60 mg/kg bw/day showed pilo-erection of a moderate grade and hunched posture.This isolated finding was considered to be incidental and of no toxicological significance
No such effects were detected in male animals treated with 60 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day
-Functional performance.
There were no toxicologically significant changes in functional performance.
Males treated with 60 and 120/90 mg/kg bw/day showed a statistically significant reduction (p<0.05) in forelimb grip strength. The intergroup difference was confined to one out of the three tests and in the absence of any clinical signs of neurotoxicity, the intergroup difference was considered not to be of toxicological importance.
- Sensory reactivity:
There were no treatment-related changes in sensory reactivity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- For males treated at 120/90 mg/kg bw/day: both body weight relative and absolute organ weights were statistically significantly higher than controls for the adrenals (p<0.01), epididymides (p<0.05), kidneys (p<0.05), liver (p<0.01), spleen (p<0.01) and testes (p<0.05). All individual kidney weights, both absolute and body weight relative were within background control ranges. One male absolute adrenal weight exceeded the background control range but the same is also true for the control males, and once values at 120/90 mg/kg bw/day were adjusted for body weight, all values were within background control normal ranges. For the liver, spleen and testes, All absolute values and the majority of individual body weight relative values were within in background control range.
- At 120/90 mg/kg bw/d, One absolute epididymis weight exceeded the background control range. When adjusted for body weight, 6/12 epididymides weights were outside the background control range, however the significance of this finding is equivocal as 2/12 control values were also outside background control ranges.

- For females treated at 120/90 mg/kg bw/day: both absolute and body weight relative pituitary weights were statistically significantly lower than controls. However, only 2/9 individual absolute weights and 3/9 individual body weight relative pituitary weights were below the background range. One control value for both absolute and body weight relative pituitary weights was also below the background normal range.

- In the absence of any histopathological correlates, it is considered that these organ weight findings are a result of natural variation, rather than any true effect of toxicology. This conclusion is supported by the fact that a large percentage of the individual values at 120/90 mg/kg bw/day are within the background control range.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence, type and distribution of macroscopic findings observed at terminal necropsy of surviving animals did not indicate any effect of treatment at dosages of 30, 60 or 120/90 mg/kg bw/day. One control male was observed to have a small and flaccid right teste and a small right epididymis, one control female had only one ovary present at necropsy. One female dosed with 60 mg/kg bw/day had an enlarged liver, and one female dosed with 120/90 mg/kg bw/day had pale lungs.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- Premature decedents
Females 94 and 90 (120 mg/kg bw/day): The animals were killed for welfare reasons on Day 2 and 5 respectively after showing gasping/laboured respiration and general malaise. At necropsy the gastrointestinal tract exhibited gaseous distension. At the microscopic examination there was marked necrosis in the trachea and death is considered to be related to this change, which possibly reflects esophageal reflux.

Female 91 (120 mg/kg bw/day) was killed for welfare reasons on Day 6 with similar clinical signs and necropsy changes but with no notable changes were apparent at histopathology.

After reduction of the dose level there were no further deaths until Day 51, when Female 87 (120/90 mg/kg bw/day) died during the bleed procedure. There were no notable changes were apparent at necropsy or histopathology and this death was considered most likely to be incidental and unrelated to treatment.


Histopathological examination of tissues from the control and 120/90 mg/kg bw/day animals did not reveal any findings considered to be related to treatment with the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis: Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 30, 60 or 120/90 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

mortality

Key result

Critical effects observed:

no

Analytical verification

Method validation

The specificity of the analytical method was demonstrated by the absence of a peak at the characterisation retention time for test substance in the control sample chromatogram.

The calibration data for the calibration standards of the test item was found to have a linear correlation within the calibration range of 0.05045 mg/mL to 0.2018 mg/mL. The R2 fit of the calibration curve to the data was 0.9999, and was considered to be acceptable.

Method accuracy (recovery) and precision were confirmed. A mean recovery value of 96% (CV=2.97%, n=5) was obtained for 2.5 mg/mL and 98% (CV=0.678%,n=5) was obtained for 50 mg/mL.

The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.

Homogeneity and stability of dose formulations

The homogeneity and stability of test substance is Arachis Oil formulations was assessed with respect to the level of concentration at nominal concentrations of 2.5 mg/mL and 50 mg/mL. Homogeneity was confirmed at the initial stability time point. The mean analyzed concentration for the nine samples remained within 10% of the initial time zero value and the variation was less than 10%. The homogeneity and stability was confirmed for the test substance in Arachis oil formulations at nominal concentrations of 2.5 and 50 mg/mL when stored at ambient temperature for 4 hours.

Test substance formulations

The results indicate that the prepared formulations were within 91 - 102% of the nominal concentration.

Summary of the results from the 14day repeated dose oral (gavage) range-finding toxicity study in the rat.

Administration of the test substance in the rat for fourteen consecutive days resulted in all animals of either sex treated with 200 mg/kg bw/day being found dead on Day 2, after receiving two consecutive daily doses of the test item. No clinical signs were observed prior to death. Macroscopic examination revealed reddened glandular regions of the stomach for all mles and one female. At 100 mg/kg bw/day any effects seen were primarily due to one male animal showing body weight loss and clinical signs between days 1 to 4 of treatment. One additional male exhibited noisy respiration on Day 6 only. No further effects were observed in the remaining one male and three females. Based on the findings of this study, 200 mg/kg bw/day was deemed too high for continued dosing, due to deaths at this dose level. Therefore, the recommended dose levels for the main study are 30, 60 and 120 mg/kg bw/day.

Conclusions:

The oral administration of the test substance to rats by gavage at dose levels of 30, 60 and 120 mg/kg bw/day resulted in the premature death of three females within the first week of treatment at 120 mg/kg bw/day. Microscopic examination revealed marked necrosis of the trachea due to possible esophageal reflux in two of these animals. This dose level was reduced to 90 mg/kg bw/day on Day 11 of study and there were no further treatment-related deaths.
Based on the findings in this study the No Observed Adverse Effect Level (NOAEL) was considered to be at least 90 mg/kg bw/day for systemic toxicity in both males and females.
The substance is classified as a Cat 1A skin corrosion and a Cat 1 serious eye damage (corrosion). Observations of this nature are common following the oral administration of a corrosive or irritant test item formulation and are considered not to represent an adverse systemic effect of treatment. Therefore, these effects do not, by themselves or together, indicate “significant” toxicity in relation to Specific Organ Toxicity – Repeated Exposure (STOT RE) classification in GHS, but a localized effect as a result of dosing. Since these observations in the substance study are considered not to support STOT RE classification, there is no need for this classification.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

90 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 60 and 120 mg/kg bw/day. Following the premature termination of one female at 120 mg/kg bw/day on Day 2 of treatment, an additional female from the spare animals on this study was included into the 120 mg/kg bw/day dose group on Day 2 to maximise the reproductive assessment. All procedures were therefore performed relative to its dose start date. A further two females at this dose level were killed in extremis during the first week of treatment, it was decided to reduce the dose level to 90 mg/kg bw/day on Day 11 of the study. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same period.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.  

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4). Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively.  Any female which did not produce a pregnancy was terminated around the same time as littering females.  All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Three females were killed in extremis during the first two weeks of treatment, due to the severity of the clinical observations noted at 120 mg/kg bw/day.  Marked necrosis of the trachea was observed in 2/3 females, due to possible esophageal reflux.  The remaining female showed no notable changes. A further female at 120/90 mg/kg bw/day died during the blood sampling procedure on Day 13 post partum (Day 51 of study).  However, there was no notable changes at necropsy or histopathology.

There were no further unscheduled deaths during the study. At 120/90 mg/kg bw/day animals of either sex exhibited increased salivation and noisy respiration on a number of occasions throughout the study. Similar observations were also seen at the lower dose levels of 60 and 30 mg/kg bw/day in both sexes; albeit at a lower frequency to animals dosed with 120/90 mg/kg bw/day.

There were no toxicologically significant effects detected in the behavioral assessments. There were no toxicologically significant changes detected in the functional performance parameters measured. There were no treatment-related changes in sensory reactivity. Males treated with 120 mg/kg bw/day initially showed a reduction in body weight gains during the first two weeks of treatment.  Following the reduction of the dose level to 90 mg/kg bw/day body weight gains were generally similar to controls.  No such effects were identified in females treated with 120/90 mg/kg bw/day or animals of either sex at 30 or 60 mg/kg bw/day.  At 120/90 mg/kg bw/day food consumption for animals of either sex was slightly lower compared to controls during the first and/or second week of treatment. Food intake thereafter was comparable to controls.  No such effects were detected in animals of either sex at 60 or 30 mg/kg bw/day or for females treated with 120/90 mg/kg bw/day during gestation and lactation phases. Similar trends were noted for food conversion efficiency. Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Assessment of hematology parameters did not indicate any obvious effect of treatment for either sex at 30, 60 or 120/90 mg/kg bw/day. Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 30, 60 or 120/90 mg/kg bw/day. There were no treatment-related abnormalities detected at necropsy.

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 30, 60 or 120/90 mg/kg bw/day. There were no toxicologically significant changes detected in the organ weights measured.  

Histopathological examination did not reveal any findings considered to be related to treatment with the test item in animals surviving until the end of the treatment period.  

The three animals that died during the first week of treatment, 2/3 females showed marked necrosis in the trachea and the deterioration of health was considered to be related to this change, which possibly reflects esophageal reflux. With no notable changes apparent for Female 91 or Female 87 which died during the latter stage of the study.

The oral administration of BOPA to rats by gavage at dose levels of 30, 60 and 120 mg/kg bw/day resulted in the premature death of three females within the first week of treatment at 120 mg/kg bw/day. Microscopic examination revealed marked necrosis of the trachea due to possible esophageal reflux in two of these animals. This dose level was reduced to 90 mg/kg bw/day on Day 11 of study and there were no further treatment-related deaths.

The substance is classified as a Cat 1A skin corrosion and a Cat 1 serious eye damage (corrosion). Observations of this nature are common following the oral administration of a corrosive or irritant test item formulation and are considered not to represent an adverse systemic effect of treatment. Therefore, these effects do not, by themselves or together, indicate “significant” toxicity in relation to Specific Organ Toxicity – Repeated Exposure (STOT RE) classification in GHS, but a localized effect as a result of dosing. Since these observations in the substance study are considered not to support STOT RE classification, there is no need for this classification. Since no adverse effects have been identified at 90 mg/kg bw/day or below, this dose level for either sex, the NOAEL remain 90 mg/kg bw/day per study conclusion.

Repeated dose toxicity - inhalation:

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

Repeated dose toxicity - dermal:

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure.

Justification for classification or non-classification

Based on the results in the combined repeated dose toxicity study with reproductive/developmental screening (according to OECD guideline 422), the test substance should not be classified for Specific Target Organ Toxicity Repeated Exposure.