Registration Dossier

Administrative data

Description of key information

Walking track analysis/ neurohistology (rat): negative

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on neurotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on neurotoxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
neurotoxicity, other
Remarks:
acute
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given
Principles of method if other than guideline:
Lewis rats of different ages were treated by an intrafascicular injection into the posterior tibial nerve fascicle proximal to the trifurcation. A functional assessment by serial walking track analysis and a morphologic assessment by neurohistology were made.
GLP compliance:
no
Species:
rat
Strain:
Lewis
Sex:
not specified
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: 4-day, 3-week, and adult rats
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- no data

Route of administration:
other: intrafascicular injection
Vehicle:
physiological saline
Details on exposure:
EXPOSURE:
- Each rat received a posterior tibial nerve intrafascicular injection using either 10% ammonium sulfate (n = 24 per age group), 0.5% bupivacaine (n = 18 per age group), 0.9% saline (n = 18 per age group), or 5% phenol (n = 18 per age group).

VOLUME:
- proportional to nerve size.
0.01 mL in 4-day-old
0.02 mL in 3-week-old rats
0.03 mL in adult rats

CONTROLS:
- Sham animals underwent sciatic nerve exposure without injection.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
single treatment
Frequency of treatment:
Once
Dose / conc.:
10 other: %
No. of animals per sex per dose:
in total 216 rats: 18 rats/age/agent
Control animals:
yes, sham-exposed
Details on study design:
- Post-exposure recovery period: 8 weeks:
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No data

OPHTHALMOSCOPIC EXAMINATION: No data

OTHER:
- All animals were followed for 8 weeks with serial walking track analyses to assess functional recovery (see below).
Specific biochemical examinations:
NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No data

CHOLINESTERASE ACTIVITY: No data

Neurobehavioural examinations performed and frequency:
- Using serial walking track analyses, elongation and subsequent normalization of the experimental footprint length (EPL) have been shown to correlate with posterior tibial nerve injury and recovery.
- A print length factor (PLF) was calculated to compare the normal left print length (NPL) to the EPL as follows : PLF = EPL - NPL/NPL.
- At 2, 4, and 8 weeks postoperatively, one third of the animals (n = 6) from each experimental group were sacrificed and their posterior tibial nerves
harvested for light microscopy to assess the extent of histologic damage.
Sacrifice and (histo)pathology:
- Time point of sacrifice: 2, 4, and 8 weeks postoperatively
- Number of animals sacrificed: postoperatively, one third of the animals (n = 6)
- Tissues evaluated:
- Other: sections of posterior tibial nerves were investigated by light microscopy to assess the extent of histologic damage
- Type of staining: Toluidine blue
- Methodology of preparation of sections:
- No data
- Number of animals evaulated from each sex and treatment group: one third
Other examinations:
No other examinations performed.
Positive control:
No positive control
Statistics:
No statistics were perfoemd.
Details on results:
CLINICAL SIGNS AND MORTALITY
- No data

BODY WEIGHT AND WEIGHT GAIN
- No data

OPHTHALMOSCOPIC EXAMINATION
- No data

BIOCHEMISTRY
- No data

NEUROBEHAVIOUR
- Walking track analysis:
• No functional abnormality was detected in any animal receiving AMS, or saline injection.

GROSS PATHOLOGY
- No data

NEUROPATHOLOGY
- Histology:
• Saline injection did not cause histologic abnormality in any age group.
• AMS caused no abnormality in the 4-day-old or 3-week-old pups; however, in the adult group, it caused mild focal injury, which recovered completely by 8 weeks postinjection.

OTHER FINDINGS
- No data
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Species:
rat

Additional information

Lewis rats of different ages (4 -day, 3 -week, and adult rats, 18 animals per age) were treated by an intrafascicular injection of ammonium sulfate (AMS, 10 %) into the posterior tibial nerve fascicle proximal to the trifurcation. A functional assessment by serial walking track analysis and a morphologic assessment by neurohistology were made (Hertl 1998). At 2, 4, and 8 weeks postoperatively, one third of the animals from each experimental group were sacrificed and their posterior tibial nerves harvested for light microscopy to assess the extent of histological damage. Concerning walking track analysis, no functional abnormality was detected in any animal receiving AMS, or saline injection. AMS caused no histological abnormalities in the 4-day-old or 3-week-old pups; however, in the adult group, it caused mild focal injury, which recovered completely by 8 weeks postinjection. Ammonium sulfate did not cause irreversible histological or functional damage in the rat nerve model.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179.