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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data is from peer reviewed publication

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Evaluation of the Potential Effects of Ingredients Added to Cigarettes. Part 4: Subchronic Inhalation Toxicity
Author:
Vanscheeuwijck PM, Teredesai A, Terpstra PM, Verbeeck J, Kuhl P, Gerstenberg B, Gebel S, Carmines EL
Year:
2002
Bibliographic source:
Food Chem Toxicol. 2002 Jan; 40(1): 113-31
Reference Type:
publication
Title:
Evaluation of the Potential Effects of Ingredients Added to Cigarettes . Part 1 : Cigarette Design, Testing Approach and Review of Results
Author:
E. L. CARMINES
Year:
2002
Bibliographic source:
Food and Chemical Toxicology, Volume 40, Issue 1, January 2002, Pages 77–91

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Principles of method if other than guideline:
Repeated dose oral toxicity study was performed for the test chemical Methyl anthranilate using cigarrette smoke as per the OECD guideline 413.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl anthranilate
EC Number:
205-132-4
EC Name:
Methyl anthranilate
Cas Number:
134-20-3
Molecular formula:
C8H9NO2
IUPAC Name:
methyl 2-aminobenzoate
Details on test material:
- Name of test material: Methyl anthranilate
- Molecular formula: C8H9NO2
- Molecular weight: 151.1641 g/mol
- Substance type: Organic
- Physical state: No data available
- Impurities: No data available

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Germany.
- Age at study initiation: 6 weeks (5 weeks old when bought and 8 days of acclimatization)
- Weight at study initiation: mean weight of male rats – 188g and female rats -150g
- Fasting period before study:
- Housing: Two rats of the same sex were housed together in polycarbonate cages supplied with sterilized softwood granulate bedding material
- Diet (e.g. ad libitum): A sterilized, fortified pellet diet (Altromin, standard rodent diet, Germany) ad libitum
- Water (e.g. ad libitum): heat-treated tap water (>68 °C for at least 10 min) ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1°C
- Humidity (%): 58 ± 4%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:
other: Inhalation: Smoke
Type of inhalation exposure:
nose only
Vehicle:
not specified
Remarks on MMAD:
MMAD / GSD: MMAD: 0.48 µm
GSD: 1.7
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Type IC88 exposure chambers in glass tubes matching their size
- Method of holding animals in test chamber: No data available
- Source and rate of air: No data available
- Method of conditioning air: No data available
- System of generating particulates/aerosols: No data available
- Temperature, humidity, pressure in air chamber:
Temp: 21.6 -23.3 °C
Humidity: 50 ± 4%
- Air flow rate: No data available
- Air change rate: No data available
- Method of particle size determination: No data available
- Treatment of exhaust air: No data available

TEST ATMOSPHERE
- Brief description of analytical method used: No data available
- Samples taken from breathing zone: No data available

VEHICLE (if applicable)
- Justification for use and choice of vehicle: No data available
- Composition of vehicle: No data available
- Type and concentration of dispersant aid (if powder): No data available
- Concentration of test material in vehicle: No data available
- Lot/batch no. of vehicle (if required): No data available
- Purity of vehicle: No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Several smoke constituents
were determined at designated time intervals . Smoke samples were collected from
within the exposure chambers at the breathing zone of the rats . The analytical methods used to determine TPM, CO, aldehydes (formaldehyde, acetaldehyde and acrolein), nicotine, nitrogen oxides, and particle size distribution as well as those for temperature and relative humidity in the exposure chambers were performed
Duration of treatment / exposure:
90 days
Frequency of treatment:
6 h/day, 7 days/week, for 90 days (The inhalation period began with a 3-day dose and tube adaptation period, i .e ., 1/4, 1/2, and 3/4 of the final daily exposure duration on days 1 (1 .5 hours exposure), 2 (3 hours 0 exposure), and 3 (4 .5 hours exposure), respectively.)
Doses / concentrations
Remarks:
Doses / Concentrations:
Low level: < 1 ppm (< 1 mg/L) High level: < 1 ppm (< 1 mg/L) Dose: 150 µg/L
Basis:
other: The low level of each ingredient in the groups approximated the typical use levels considered to be reflective of those used in modern cigarettes and the high levels were 1 .5 or 3 times the low levels .
No. of animals per sex per dose:
Total: 68
Control group: 14 male and 14 female rats
Reference Cigarette group: 10 male, 10 female
Exposure Group: 10 male, 10 female
Control animals:
yes, concurrent vehicle
Details on study design:
No data available
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality: Atleast once on each day
General conditions: shortly after the daily exposure
- Cage side observations checked in table [No.?] were included. Mortality/Moribundity, Detailed checks of the general condition and behavior of the rats were performed on 2 to 4 rats per group per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once/week

BODY WEIGHT: Yes
- Time schedule for examinations: Once/week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: performed on all rats before the start of the inhalation period
and on all rats in the sham, control, high ingredient level, and 1 R4F groups at the end of
the inhalation period .
- Dose groups that were examined: Control, reference and test groups were examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: Yes, of sodium pentobarbital
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. The usine was checked for the presence of urinary nicotine metabolites

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, At necropsy, the rats were killed by exsanguination after pentobarbital anesthesia . An external examination of the carcass was conducted to determine the presence of gross lesions. All rats were then completely dissected and examined for gross lesions of the internal tissues and organs. Weights of lungs with larynx and trachea, liver, heart, adrenal glands, and kidneys were recorded.

HISTOPATHOLOGY: Yes, Lungs with larynx and trachea were removed together and fixed with EAFS (ethanol-acetic acid-formaldehyde-saline solution) solution by instillation at 20 cm water pressure. Testes were fixed in Bouin's solution, sternum in Schaffer's solution, eyes in Davidson's solution, and all other organs in 10% buffered formaldehyde solution. Histological sections were prepared for the nose according to the method of Young and for the larynx according to Lewis. The trachea was cut transversally (near the larynx) as well as frontally (at the bifurcation). For the left lung, 1 frontal section passing through the main bronchus was prepared, and for the right lung, I frontal section passing through a maximum number of lobes was prepared. All sections were stained with hematoxylin-eosin (HE). Sections from nose level 1, tracheal ring and bifurcation, and lungs were additionally stained with alcian blue/periodic acid Schiff's reagent and liver sections with oil-red orange stain. The laryngeal epithelial thickness at the floor of the larynx was also determined morphometrically. A veterinary pathologist with experience in cigarette smoke-related changes in the respiratory tract of rodents read the slides in a blind manner. Histopathological findings were scored according to a defined severity scale from 0 to 5. Mean severity scores were calculated based on all rats in a group.
Other examinations:
Biomonitoring: Respiratory frequency, tidal volume, and respiratory minute volume were determined on 10 rats/sex/group by whole body plethysmography to provide an estimate of the inhaled dose of cigarette smoke. Steady state concentrations of blood carboxyhemoglobin (HbCO) were determined in 5 rats/sex/group according to the method of Klimisch, once during the inhalation period.
Statistics:
For continuous data, the one-way analysis of variance was used for the overall comparison followed by a pairwise comparison (Duncan), or the t-test modified according to Welch (after the post-inhalation period) . For ordinal data the generalized Cochran-Mantel-Haenszel test (CMH) was used for overall comparison followed a pairwise comparison where appropriate. Results were evaluated for statistical significance at p<0 .05 without adjustment for multiple testing . Statistically significant results were considered as explorative indicators rather than confirmatory evidence of an effect except in cases where the parameters evaluated revealed a consistent, treatment-related response that was biologically meaningful.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
Clinical signs and mortality:
Clinical signs: Exposure and smoke-related findings were increased irritability, decreased reflexes, Harderian gland secretion, and moist or wet fur. At the end of the inhalation period, smoke-exposure related changes were yellow discoloration of the fur, incidental occurrence of decubital ulcers on the paws.

Mortality: No smoke related mortality was observed

Body weight and weight gain:
The body weight of both male and female rats in all groups increased throughout the inhalation period. By the end of the inhalation period, the body weight gain for all male smoke-exposed rats was reduced by 20 to 28% compared to the sham group. This reduction is considered to be related to MS-toxicity and irritation-dependent stress.

Food consumption and compound intake: The addition of groups of ingredients to the cigarettes did not affect the food consumption of the rats

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: The ophthalmological examination did not reveal any ingredient-related effect or alteration in eyelids with conjunctivae, cornea, iris, lens, vitreous body, or retina.

Haematology: No statistically significant differences were observed between the test and control groups for any of the hematological parameters at the end of the inhalation period, except for a higher differential lymphocyte count in male rats exposed to smoke from Ingredient Group 1 (low level) cigarettes and for a lower differential neutrophil count in the group of female rats exposed to smoke from Ingredient Group 3 (high level cigarettes). These differences, however, are considered to be isolated findings without biological significance.

Clinical chemistry: The differences of clinical chemistry parameters between the test and control groups were inconsistently spread over both sexes and over the different exposure groups and were considered to be incidental

Urinanalysis: The urinary nicotine metabolite profiles were nearly the same for all groups; however, the excretion of trans-3'-hydroxycotinine was slightly higher in male rats than in female rats

Neurobehaviour: No data

Organ weights: No significant differences for either absolute or relative weight were seen between the test and control groups, with the exception of the thymus. Male rats in the test groups exhibited a less pronounced decrease in thymus weight than those in the control group At the end of the post-inhalation period, thymus atrophy was no longer observed in any of the rats (thymus weight and size equal to sham).

At the end of the inhalation period, the absolute and relative weight of lungs with larynx and trachea for all rats was statistically significantly higher in the smoke-exposed groups than in the sham. This smoke effect Is equal for the control and the test cigarette smoke-exposed groups (male and female) .

Gross pathology: No remarkable differences in in-life observations or gross pathology between test and control groups was noted

Histopathology:
The basic smoke-related histopathological effects, which were more pronounced in the upper respiratory tract than in the lower respiratory tract, were hyperplasia and squamous metaplasia of the respiratory epithelium, squamous metaplasia and atrophy of the olfactory epithelium, and accumulation of pigmented alveolar macrophages . There were no relevant qualitative or quantitative differences in findings in the respiratory tract of the rats exposed to the smoke from the control and test cigarettes.

Effect levels

Dose descriptor:
NOAEC
Effect level:
< 1 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The biological activity of the main stream smoke was not affected by the addition of the chemical

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Biomonitoring:

Respiratory Physiology: The respiratory rates and tidal volumes obtained for the smoke-exposed rats were the same for both test and control groups ; the respiratory minute volumes were at least as high in the test groups as in the control group.

 

Carboxyhemoglobin: Smoke from test cigarettes supplemented with low and high levels of ingredient had no effect on steady-state carboxyhemoglobin concentrations in male and female rats compared to smoke-exposed controls.

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse effect concentration (NOAEC) for the test compound Methyl anthranilate is found to be <1 ppm (< 1 mg/L).
Executive summary:

Subchronic repeated dose oral toxicity study was performed for the test chemical Methyl anthranilate as per the OECD guideline 413 using cigarrette smoke containing <1 mg/L test compound depicted as low and high level. The low level of each ingredient in the groups approximated the typical use levels considered to be reflective of those used in modern cigarettes and the high levels were 1 .5 or 3 times the low levels. Male and female Sprague Dawley rats were nose-only exposed either to fresh air (sham) or to diluted mainstream smoke from the test, the control, or the Reference Cigarette 1R4F at a concentration of 150 pg total particulate matter/I for 90-days, 6 h/day, 7 days/week . A 42-day post-inhalation period was included to evaluate reversibility of possible findings.

 

There were no remarkable differences in in-life observations or gross pathology between test and control groups. An increase in activity of liver enzymes, known to be due to the high smoke dose, revealed no toxicologically relevant differences between the test and control groups . No toxicological differences were seen between the test and control groups for smoke-related hematological changes, such as a decrease in total leukocyte count . The basic smoke-related histopathological effects, which were more pronounced in the upper respiratory tract than in the lower respiratory tract, were hyperplasia and squamous metaplasia of the respiratory epithelium, squamous metaplasia and atrophy of the olfactory epithelium, and accumulation of pigmented alveolar macrophages . There were no relevant qualitative or quantitative differences in findings in the respiratory tract of the rats exposed to the smoke from the control and test cigarettes.

 

The addition of the test chemical commonly used ingredients added to cigarettes did not increase the biological activity of the mainstream smoke, even at the exaggerated levels used. Hence, The No Observed Adverse effect concentration (NOAEC) for the test compound Methyl anthranilate is found to be <1 ppm.

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