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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 March 2013 to 30 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with intertnational guidelines (OECD 412) and in compliance with the UK Good Laboratory Practive Regulations. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
including 14 day recovery period
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Identification: Allylamine

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 (UK) Ltd
- Age at study initiation: 53 to 60 days old
- Weight at study initiation: 264 to 318g males; 177 to 224g females
- Fasting period before study: none
- Housing: caged in 5's
- Diet (e.g. ad libitum): ad libitum except during exposures
- Water (e.g. ad libitum):ad libitum except during exposures
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: March 2013 To: May 2013

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose/head only
Remarks:
reported as snout only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through snout only exposure chamber
- Method of holding animals in test chamber: Polycarbonate restraining tubes with their snouts protruding
- Source and rate of air: in-house compressed air system – breathing quality: 58L/min (control); 40 L/min (test groups)
- Method of conditioning air: not stated
- System of generating particulates/aerosols: Metered supply of test material onto small sintered glass vapouriser
- Temperature, humidity, pressure in air chamber: Essentially atmosperic pressure. Temp/hum not stated
- Air flow rate: 58L/min (control); 40 L/min (test groups)
- Air change rate: not stated
- Method of particle size determination: not applicable
- Treatment of exhaust air: filtered locally

TEST ATMOSPHERE
- Brief description of analytical method used: samples in trapping solvent, followed by HPLC analysis. minimum of 3 samples/group/exposure
- Samples taken from breathing zone: representative animal level
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The achieved levels were 100, 103 and 100% of the target concentrations for Groups 2, 3 and
4 respectively.
Duration of treatment / exposure:
6 hours per day for 28 days followed by 14 day off-treatment period for recovery animals
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 ppm (analytical)
Dose / conc.:
10.5 ppm (analytical)
Dose / conc.:
20.3 ppm (analytical)
No. of animals per sex per dose:
5M/5F per group dosed for 28 days.
5M5F in Control and High dose, dosed for 28 days followed by 14 day recovery period
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were dosed for 28 days at dose concentrations of 5, 10 and 20 ppm, 6 hours per day. Dose levels were selected from a preliminary study (Horrell, 2013a).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pre-exposure; at return to home cage; at end of work day

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Consumption recorded weekly, but per kg not calculated

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

WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Near end of 4 week dosing period, near end of recovery period (specified parameters only)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all 5 main study animals per group/sex, then all recovery animals for recovery sampling only
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Near end of 4 week dosing period, near end of recovery period (specified parameters only)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all 5 main study animals per group/sex, then all recovery animals for recovery sampling only
- Parameters checked in table [No.?] were examined.


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Detailed physical exam and arena observations conducted weekly, including pre-trial and recovery phases. For sensory activity, motor activity and grip strength, all Low and Mid dose animals, plus recovery group animals from Control and High dose, were examined in 4th week of dosing
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other: detailed clinical observations

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Statistics:
bodyweight, using gains over appropriate study periods.
blood chemistry and haematology.
grip strength and motor activity.
organ weights, either absolute or adjusted for terminal bodyweight where appropriate.

For categorical data, the proportion of animals were analysed using Fisher’s Exact test for each treated group versus the control.
For continuous data, Bartlett’s test was applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs related to treatment with allylamine. Signs associated with the method of exposure included wet fur and red staining on the head of some animals from all groups, evident on return to the home cage. For a few animals these signs persisted until the final observation check of the day.
Mortality:
no mortality observed
Description (incidence):
There were no clinical signs related to treatment with allylamine. Signs associated with the method of exposure included wet fur and red staining on the head of some animals from all groups, evident on return to the home cage. For a few animals these signs persisted until the final observation check of the day.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Changes in bodyweight reversed after recovery period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no clear treatment related changes to food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Week 4 group mean WBC counts were significantly lower than control values for males of Groups 3 and 4 (both 0.78X control). Similar changes were not evident in females. No effects observed after 2 week recovery period.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Week 4 group mean triglycerides were significantly lower for Group 4 females (0.66X control). Mean phosphate concentration was significantly higher for Group 4 females (1.21X control), but these changes were considered unrelated to allylamine.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no evident treatment-related changes in sensory reactivity tests. Isolated minor differences in low beam activity scores were considered incidental in absence of high beam score effects, or any effects in females.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Bodyweight adjusted mean lung and bronchi weight was significantly higher for Group 4 females (1.07X). Bodyweight adjusted mean liver weight was also significantly higher for Group 4 females (1.12X). No effects observed after 2 week recovery.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed after 4 weeks of treatment, and following 2 week recovery, revealed no intergroup differences of note.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No toxicologically relevant findings were seen in the heart, lungs, liver and kidneys of Control and high dose animals at the end of 28 days exposure, and following 2 week recovery.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
20.3 ppm (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
20.3 ppm (analytical)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Reductions in White Blood Cell (WBC) counts (0.78X and 0.78X control), lymphocytes (0.80X and 0.77X control) and basophils (0.43X and 0.57X control), were evident for males after 4 weeks exposure at to 10.5 or 20.3 ppm, persisting for males previously exposed to 20.3 ppm after a 2-week recovery period (WBC, lymphocyte and basophil counts 0.86X, 0.84X and 0.9X control respectively). These differences were not evident in females and, in the absence of any associated histopathological changes, are considered to be of no toxicological importance.
Conclusion
Exposure of Sprague-Dawley rats to Allylamine, using a snout-only inhalation exposure system, for 6 hours a day for 7 consecutive days at achieved aerosol concentrations of 5.1, 10.5 and 20.3 ppm was well tolerated and did not result in any histopathological changes.
Slight reductions in bodyweight gain when compared with control were evident for males from all Allylamine exposed groups. A similar effect in females was not clear and the effect in males previously exposed to 20.3 ppm was reversed following a 2 week off-exposure recovery period. The effect on bodyweight was thus not considered to be adverse over the duration of this study, but could potentially be so on a longer study.
The No Observed Adverse Effect Level (NOAEL), is thus considered to be 20.3 ppm.
Executive summary:

The systemic toxic potential of Allylamine was assessed when administered to Sprague Dawley rats by snout-only inhalation administration for 4 weeks. Recovery from any effects was evaluated during a 2 week recovery period.

Three groups, each comprising 5 male and 5 female Sprague Dawley rats, received Allylamine at target exposures of 5, 10 or 20 ppm. A similarly constituted control group received air only, at the same operating conditions as the high dose group. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for 4 weeks, followed by a 2 week period without treatment to assess the potential for any treatment-related change to recover.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, haematology (peripheral blood), haematology (bone marrow), blood chemistry, organ weights, macropathology and histopathology investigations were undertaken.

The overall achieved aerosol concentrations were 102, 105 and 102% of the target concentration for Groups 2, 3 and 4, respectively.

There were no unscheduled deaths, treatment related clinical signs, effects on food consumption, sensory reactivity, grip strength, motor activity, blood chemistry parameters, organ weights or macroscopic or microscopic pathological findings.

Mean bodyweight gains over the 4 weeks of treatment were statistically significantly lower than control for males exposed to 5.1, 10.5 or 20.3 ppm (0.78, 0.74 and 0.75X control respectively). Mean bodyweight gain was also lower than control for females exposed to 10.5 ppm (0.76X control), however this difference did not attain statistical significance and mean bodyweight gain for females exposed to 20.3 ppm was similar to control (0.95X control). After 2 weeks of recovery mean bodyweights were similar to control (1.06X control) for males previously exposed to 20.3 ppm, but lower than control (0.72X control) for females previously exposed to 20.3 ppm.

Reductions in White Blood Cell (WBC) counts (0.78X and 0.78X control), lymphocytes (0.80X and 0.77X control) and basophils (0.43X and 0.57X control), were evident for males after 4 weeks exposure at to 10.5 or 20.3 ppm, persisting for males previously exposed to 20.3 ppm after a 2-week recovery period (WBC, lymphocyte and basophil counts 0.86X, 0.84X and 0.9X control respectively). These differences were not evident in females and, in the absence of any associated histopathological changes, are considered to be of no toxicological importance.

Conclusion

Exposure of Sprague-Dawley rats to Allylamine, using a snout-only inhalation exposure system, for 6 hours a day for 7 consecutive days at achieved aerosol concentrations of 5.1, 10.5 and 20.3 ppm was well tolerated and did not result in any histopathological changes.

Slight reductions in bodyweight gain when compared with control were evident for males from all Allylamine exposed groups. A similar effect in females was not clear and the effect in males previously exposed to 20.3 ppm was reversed following a 2 week off-exposure recovery period. The effect on bodyweight was thus not considered to be adverse over the duration of this study, but could potentially be so on a longer study.

The No Observed Adverse Effect Level (NOAEL), is thus considered to be 20.3 ppm.