3,4-dimethylbenzaldehyde

Endpoint summary

Description of key information

A NOEL of 50 mg/kg bw/day for females and of 250 mg/kg bw/day for males was established in a combined repeat dose toxicity study with reproduction/developmental toxicity screening test in rats (OECD 422, GLP).

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

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

SafePharm Laboratories Ltd., Shardlow Business Park, London Road, Shardlow, Derby, DE72 2GD, UK

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males 250-305 g, females 169-210 g.
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase, animals were transferred to similar cages on a one male:one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks (B&K Universal Ltd, Hull, UK) and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation.
- Diet: A pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Ltd., London, UK, ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±2
- Humidity (%): 55±15
- Air changes (per hr): ≥15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. The stability and homogeneity of the test material formulations were determined. The formulations were stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4°C in the dark.

VEHICLE
- Amount of vehicle: 4 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test material formulation were taken on a weekly basis and analysed for concentration of 3,4-Dimethylbenzaldehyde. The concentration of 3, 4-Dimethyl Benzaldehyde in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The test material formulations were diluted with acetonitrile to give a final, theoretical test material concentration of approximately 0.05 mg/mL. Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.05 mg/mL. For homogeneity determinations, the test material formulations were mixed thoroughly and. samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate. For stability determinations, the test material formulations were sampled and analysed initially and then after storage at approximately +4°C in the dark for fourteen days. The results indicate that the analysed formulations were within acceptable limits for the purpose of this study.

Duration of treatment / exposure:

up to forty-six consecutive days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
50, 250, 1000 mg/kg bw
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 preliminary range-finder in the rat.
- Chronological Sequence of Study: Groups of ten male and ten female animals were treated daily (from Day 1 of the study) at the appropriate dose level throughout the study (except for females during parturition where applicable). Prior to the start of treatment and once weekly, all animals were observed for signs of functional/behavioural toxicity. One day prior to pairing (Day 14), blood samples were taken from five males and five females, randomly selected from each dose group and analysed for haematological and blood chemical assessment. Blood samples were also taken at termination from five males and five females and analysed for haematological and blood chemical assessment. On Day 15, all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days. Following evidence of mating using vaginal smearing, the males were returned to their original cages and females were transferred to individual cages. On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean pup weight, clinical observations and reflexological response were also performed during this period. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically. At Day 5 post partum, all surviving females and offspring were killed and examined macroscopically.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, determined as g/animal/day
- During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded during the period of gestation. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period and for females during maturation and the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.

WATER CONSUMPTION: Water intake was observed daily by visual inspection of water bottles for any overt change.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 14 (day prior to pairing) and at termination
- Animals fasted: No
- How many animals: 5 males and 5 females of each test and control group
- Parameters examined: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices: [mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)], Total leucocyte count (WBC), Differential leucocyte count: [neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Reticulocyte count (Retic; cresyl blue stained slides were prepared but reticulocytes were not assessed)], Platelet count (PLT), Prothrombin time (CT), Activated thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 14 (day prior to pairing) and at termination
- Animals fasted: No
- How many animals: 5 males and 5 females of each test and control group
- Parameters examined: Urea, Glucose, Total protein, Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium(Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine, Total cholesterol, Total bilirubin.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females per dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Dose groups that were examined: all
- Battery of functions tested: behavioural assessment, functional performance tests (motor activity, Forelimb/Hindlimb Grips Strength), Sensory reactivity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS: The following organs, removed from the five selected males and parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation. The reproductive organs were weighed from all animals that were killed at the end of the study: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus.

HISTOPATHOLOGY: Yes, samples of the following tissues from five males and five females from control and 1000 mg/kg/day dose group and those animals dying during the study: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Eyes, Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Mammary gland, Muscle (skeletal), Pancreas, Oesophagus, Rectum, Salivary glands (submaxillary), Sciatic nerve, Skin (hind limb), Spinal cord (cervical, thoracic and lumbar), Spleen, Stomach, Thyroid/parathyroid, Trachea, Thymus, Urinary bladder, Vagina. The following tissues were examined from all animals of control and 1000 mg/kg/day dose group: Coagulating gland, Epididymides, Ovaries, Pituitary, Prostate, Seminal vesicles, Testes, Uterus/Cervix. Since there were indications of treatment-related changes in the stomach, examination was subsequently extended to include similarly prepared sections of the stomach from five selected males and females from the 250 and 50 mg/kg/day dose groups.

Statistics:

Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test. The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.

Details on results:

MORTALITY: There were no deaths related to treatment. One female treated with 250 mg/kg/day was killed in extremis on gestation Day 24, following difficulties encountered during parturition. In isolation and in the absence of any histopathological correlates, this death was considered unrelated to treatment.

CLINICAL SIGNS: No clinically observable signs of toxicity were detected. Treatment related effects were confined to sporadic episodes of increased salivation detected immediately after dosing for animals of either sex treated with 1000 mg/kg/day from Day 4. Incidents of generalised red/brown staining and wet fur (males only) were also detected for these animals, with red/brown staining also detected for females treated with 250 mg/kg/day. In addition incidents of noisy respiration were detected for seven males and three females treated with 1000 mg/kg/day and for one male and one female treated with 250 mg/kg/day. These observations are often reported following the oral administration of a slightly unpalatable or irritant test material formulation, and in isolation, are considered to be of no toxicological importance. The interim death female treated with 250 mg/kg/day displayed pilo-erection, tip-toe gait and hunched posture during parturition, the severity of these observations subsequently led to the female being killed in extremis. The observations detected were due to difficulties encountered during parturition. As this was an isolated incident and in the absence of a dose related response this death was considered unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN: No adverse effect on bodyweight change was detected for treated males in comparison to controls throughout the treatment period, or for females throughout the maturation, gestation or lactation phases of the study.

FOOD CONSUMPTION: A statistically significant reduction in dietary intake was evident for females treated with 1000 mg/kg/day during lactation when compared to controls. No adverse effect on dietary intake was detected for males throughout the treatment period, or for females during maturation and gestation.

WATER CONSUMPTION: Daily visual inspection of water bottles revealed no intergroup differences.

HAEMATOLOGY: No treatment-related haematological changes were detected prior to mating or at termination.

CLINICAL CHEMISTRY: No treatment-related changes were detected in the blood parameters measured. A statistically significant increase in albumin/globulin ratio was detected for males treated with 1000 mg/kg/day at Day 14 and termination. The apparent increase was, in reality, due to a single low control value, and in the absence of related changes in levels of total protein and albumin, was considered unrelated to test material toxicity. Males treated with 1000 mg/kg/day also showed a statistical significant decrease in levels of calcium ions and inorganic phosphate at Day 14. As decreases in these parameters were not detected at the terminal bleeds these changes were considered to be incidental and not related to test material toxicity. Males of this treatment group at termination also showed a significant increase in alkaline phosphatase. The increase in alkaline phosphatase was of minimal statistical significance, and was a consequence of a single low value in the controls therefore, was considered unrelated to test material toxicity. A statistically significant decrease in plasma urea was detected for males treated with 1000 or 250 mg/kg/day. The significance was p<0.05 and p
NEUROBEHAVIOUR: Weekly open field arena observations did not reveal any treatment-related effects during the study. All inter and intra group variations in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance. No treatment-related effects were detected in the functional performance parameters investigated. Statistical analysis of the data did not reveal any significant inter group differences. No treatment-related effects were detected in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be result of normal variation for rats of the strain and age used and were of no toxicological importance.

ORGAN WEIGHTS: No treatment related effects were detected. Liver weights relative to terminal bodyweight were elevated for animals of either sex treated with 1000 mg/kg/day. In the absence of relating histopathological evidence to suggest damage or impaired function of the liver, this finding was considered unrelated to test material toxicity.

GROSS PATHOLOGY: The incidental macroscopic observations detected included hydronepherosis of the right kidney for a single male treated with 1000 mg/kg/day. One male treated with 250 mg/kg/day had dark contents of the bladder; and one male treated with 50 mg/kg/day had a red mass. This mass was approximately 5mm by - 10mm in size and attached to the fat surrounding the epididymis. One control male had crystalline solids in the bladder, the bladder was also distended. All these findings were considered incidental, and in the absence of a dose related response, were considered unrelated to treatment. One control male had a damaged left eye, this was a physical injury that occurred during the necropsy procedure and so was unrelated to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC: Acanthosis, occasionally with associated hyperkeratosis, was observed in relation to treatment in the forestomach among animals of either sex treated with 1000 mg/kg/day. Such changes were also observed for one female treated with 250 mg/kg/day and also in the premature death animal from this treatment level. Although such changes in the forestomach do occasionally occur spontaneously in untreated laboratory maintained rats, a relationship to treatment at this dose level cannot be reliably excluded. There was one unscheduled death during the course of the study. There was peripheral inflammation of the uterus.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Histopathology of the stomach: Acanthosis, occasionally with associated hyperkeratosis was observed in relation to treatment in the forestomach among animals treated with 1000 mg/kg/day.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Histopathology of the stomach: Acanthosis, occasionally with associated hyperkeratosis was observed in relation to treatment in the forestomach among females treated with 1000 mg/kg/day and 250 mg/kg/day.

Critical effects observed:

not specified

Conclusions:

The oral administration of the test substance to rats by gavage, at dose levels of 50, 250 and 1000 mg/kg/day, resulted in treatment-related effects detected in the stomach for animals of either sex treated with 1000 mg/kg/day and for females treated with 250 mg/kg/day. The No Observed Effect Level (NOEL) for treatment-related changes was considered to be 250 mg/kg/day for males and 50 mg/kg/day for females.

Executive summary:

In a GLP compliant combined repeat dose toxicity study with reproduction/developmental toxicity screening test, performed according to OECD Guideline 422, the test substance was administered by gavage to three groups each of ten male and ten female Sprague-Dawley rats, for up to forty-six consecutive days, at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400). Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected non-recovery males and females from each dose group, and again at termination. Extensive functional observations were performed on five selected parental males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post partum. Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination, organ weights and histopathological evaluation of selected tissues was performed. No treatment-related effects were observed on mortality, clinical signs, behavioural assessments, functional performance tests, sensory reactivity assessments, bodyweights, food consumption of males, water consumption, haematology, blood chemistry, macroscopic observations at necropsy, organ weights. Females treated with 1000 mg/kg/day showed a slight reduction in food consumption during lactation when compared to controls, but no adverse effect on dietary intake was detected for females during the maturation or gestation phases of the study. Histopathological examination revealed a treatment­related change in the stomach: acanthosis, occasionally with associated hyperkeratosis, was observed in relation to treatment in the forestomach among animals of either sex treated with 1000 mg/kg/day. Such changes were also observed for one female treated with 250 mg/kg/day and also in the premature death female animal from this treatment level. In conclusion, the oral administration of the test substance to rats by gavage, at dose levels of 50, 250 and 1000 mg/kg/day, resulted in treatment-related effects detected in the stomach for animals of either sex treated with 1000 mg/kg/day and for females treated with 250 mg/kg/day. The No Observed Effect Level (NOEL) for treatment-related changes was considered to be 250 mg/kg/day for males and 50 mg/kg/day for females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-compliant guideline study, klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a GLP compliant combined repeat dose toxicity study with reproduction/developmental toxicity screening test, performed according to OECD Guideline 422, the test substance was administered by gavage to three groups each of ten male and ten female Sprague-Dawley rats, for up to forty-six consecutive days, at dose levels of 50, 250 and 1000 mg/kg/day (Safepharm Laboratories Ltd., 2007). The dose levels were based on the findings in a dose range finding study (Safepharm Laboratories Ltd., 2007). A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400). Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected non-recovery males and females from each dose group, and again at termination. Extensive functional observations were performed on five selected parental males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post partum. Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination, organ weights and histopathological evaluation of selected tissues was performed. No treatment-related effects were observed on mortality, clinical signs, behavioural assessments, functional performance tests, sensory reactivity assessments, bodyweights, food consumption of males, water consumption, haematology, blood chemistry, macroscopic observations at necropsy, organ weights. Females treated with 1000 mg/kg/day showed a slight reduction in food consumption during lactation when compared to controls, but no adverse effect on dietary intake was detected for females during the maturation or gestation phases of the study. Histopathological examination revealed a treatment­related change in the stomach: acanthosis, occasionally with associated hyperkeratosis, was observed in relation to treatment in the forestomach among animals of either sex treated with 1000 mg/kg/day. Such changes were also observed for one female treated with 250 mg/kg/day and also in the premature death female animal from this treatment level. In conclusion, the oral administration of the test substance to rats by gavage, at dose levels of 50, 250 and 1000 mg/kg/day, resulted in treatment-related effects detected in the stomach for animals of either sex treated with 1000 mg/kg/day and for females treated with 250 mg/kg/day. The No Observed Effect Level (NOEL) for treatment-related changes was considered to be 250 mg/kg/day for males and 50 mg/kg/day for females.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only study available, GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: stomach

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity study, the substance does not need to be classified according to the Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.